Light Regulation of LHCX Genes in the Benthic Diatom Seminavis robusta

Intertidal benthic diatoms experience a highly variable light regime, which especially challenges these organisms to cope with excess light energy during low tide. Non-photochemical quenching of chlorophyll fluorescence (NPQ) is one of the most rapid mechanisms diatoms possess to dissipate excess energy. Its capacity is mainly defined by the xanthophyll cycle (XC) and Light-Harvesting Complex X (LHCX) proteins. Whereas the XC and its relation to NPQ have been relatively well-studied in both planktonic and benthic diatoms, our current knowledge about LHCX proteins and their potential involvement in NPQ regulation is largely restricted to planktonic diatoms. While recent studies using immuno-blotting have revealed the presence of light regulated LHCX proteins in benthic diatom communities and isolates, nothing is as yet known about the diversity, identity and transcriptional regulation or function of these proteins. We identified LHCX genes in the draft genome of the model benthic diatom Seminavis robusta and followed their transcriptional regulation during a day/night cycle and during exposure to high light conditions. The S. robusta genome contains 17 LHCX sequences, which is much more than in the sequenced planktonic model diatoms (4–5), but similar to the number of LHCX genes in the sea ice associated diatom Fragilariopsis cylindrus. LHCX diversification in both species, however, appears to have occurred independently. Interestingly, the S. robusta genome contains LHCX genes that are related to the LHCX6 of the model centric diatom Thalassiosira pseudonana, which are lacking in the well-studied pennate model diatom Phaeodactylum tricornutum. All investigated LHCX genes, with exception of SrLHCX6, were upregulated during the daily dark-light transition. Exposure to 2,000 μmol photons m–2 s–1, furthermore, increased transcription of all investigated LHCX genes. Our data suggest that the diversification and involvement of several light regulated LHCX genes in the photophysiology of S. robusta may represent an adaptation to the complex and highly variable light environment this benthic diatom species can be exposed to.


INTRODUCTION
Due to the complex interplay of diurnal and tidal cycles and weather conditions, the surface sediments of tidal flats experience highly variable light conditions. Nevertheless, they are very productive ecosystems thanks to the presence of biofilms dominated by benthic microalgae (microphytobenthos) and especially diatoms (Underwood and Kromkamp, 1999). The fluctuating light conditions challenge these diatoms to maximize light harvesting under low light (LL) conditions while avoiding oxidative damage to their photosynthetic apparatus under high light (HL), either by minimizing light absorbance or by the dissipation of excess light energy. Benthic diatoms possess two main strategies which are fast enough to track rapid fluctuations in light intensity, namely, vertical migration and dissipation of excess energy as heat (Lavaud and Goss, 2014;Laviale et al., 2016). Raphid pennate diatoms possess a special cell wall structure called the raphe through which mucilage is secreted, allowing the diatoms to move. Such motile diatoms, often referred to as epipelic diatoms, can form dense biofilms on fine-grained sediments (Sabbe, 1993;Ribeiro et al., 2013) and are able to position themselves within the sediment light gradient via vertical migration (Admiraal, 1984;Consalvey et al., 2004;Serôdio et al., 2006;Cartaxana et al., 2016). Dissipation of excess light energy as heat can be measured as Non-Photochemical Quenching of chlorophyll a fluorescence (NPQ). In diatoms, NPQ comprises a quickly and a slowly relaxing component (Lavaud and Goss, 2014). We will refer to the quickly relaxing component as "flexible NPQ" (Niyogi and Truong, 2013) and to the slowly relaxing component as "sustained NPQ" or NPQs (Lavaud and Goss, 2014). The capacity for flexible NPQ is mainly defined by the xanthophyll cycle (XC) pigment diatoxanthin (Dtx) produced via de-epoxidation of diadinoxanthin (Ddx) (Lavaud and Goss, 2014;Barnett et al., 2015;Goss and Lepetit, 2015;Blommaert et al., 2017), and the presence of Light-Harvesting Complex X (LHCX) proteins (Bailleul et al., 2010;Ghazaryan et al., 2016;Taddei et al., 2016Taddei et al., , 2018Lepetit et al., 2017). While the XC in benthic diatoms has been well-studied in natural communities (van Leeuwe et al., 2008;Jesus et al., 2009;Serôdio et al., 2012;Laviale et al., 2015) and more recently also using unialgal isolates Blommaert et al., 2017), our current knowledge about LHCX proteins as an NPQ regulator is mostly based on studies of planktonic diatoms (Nymark et al., 2009(Nymark et al., , 2013Büchel, 2014;Lavaud and Goss, 2014;Valle et al., 2014;Dong et al., 2015;Goss and Lepetit, 2015;Ghazaryan et al., 2016;Grouneva et al., 2016;Lepetit et al., 2017;Taddei et al., 2018Taddei et al., , 2016. The latter includes studies on the pennate model diatom Phaeodactylum tricornutum which to date has only been isolated from water samples from various coastal environments, but may have a benthic growth phase as well (De Martino et al., 2007).
Light-Harvesting Complex X proteins are closely related to the Light-Harvesting Complex Stress-Related (LHCSR) proteins that are present in most eukaryotic algae and mosses, but absent in flowering plants (Niyogi and Truong, 2013;Goss and Lepetit, 2015). Even though LHCX/LHCSR proteins are Light Harvesting Proteins, they have an energy dissipating rather than a light harvesting function (Niyogi and Truong, 2013). LHCSR proteins appear to function both as excess light sensors and quenching sites (Bonente et al., 2011;Ballottari et al., 2016). Therefore, they possess protonatable amino-acid residues that sense a low luminal pH ( pH) (Ballottari et al., 2016). A similar function as NPQ regulators has been proposed for LHCX proteins in planktonic diatoms as HL induces LHCX transcription and enhances LHCX protein content (Oeltjen et al., 2002;Nymark et al., 2009Nymark et al., , 2013Bailleul et al., 2010;Park et al., 2010;Lepetit et al., 2013Lepetit et al., , 2017Taddei et al., 2016;Hippmann et al., 2017). LHCX proteins are hypothesized to bind the XC pigments Ddx and Dtx (Beer et al., 2006;Lepetit et al., 2013) and change the supramolecular organization of antenna complexes (Ghazaryan et al., 2016). Furthermore, excess energy dissipation, mediated by LHCX proteins, was linked to a decrease in the functional absorption cross-section of photosystem II (Buck et al., 2019).
Light-Harvesting Complex X function and transcriptional regulation has been intensively studied in the diatom P. tricornutum (Nymark et al., 2009(Nymark et al., , 2013Bailleul et al., 2010;Lepetit et al., 2013Lepetit et al., , 2017Valle et al., 2014;Taddei et al., 2016;Buck et al., 2019). The P. tricornutum genome contains four LHCX genes (LHCX1-4). Of these four genes, LHCX1 is highly expressed in non-stressful light conditions, whilst additional expression upon HL exposure is low (Nymark et al., 2009;Lepetit et al., 2013;Taddei et al., 2016Taddei et al., , 2018. Its corresponding protein is consequently present in LL conditions, where it provides the diatom with a basal capacity for NPQ, localized mainly near the PSII core in P. tricornutum, to cope with sudden changes in light climate (Taddei et al., 2018). In addition, the different content in LHCX1 between different P. tricornutum ecotypes has been related to their natural variability in NPQ capacity (Bailleul et al., 2010). In (prolonged) HL conditions, both transcription of LHCX2 and LHCX3 is strongly induced (Nymark et al., 2009;Lepetit et al., 2013Lepetit et al., , 2017Taddei et al., 2016). As both proteins accumulate in concert with the de novo synthesis of Ddx + Dtx, they may provide additional Ddx/Dtx binding sites to activate NPQ in the antenna to enhance the basal NPQ provided by LHCX1 (Lepetit et al., 2013(Lepetit et al., , 2017Taddei et al., 2018;Buck et al., 2019). Indeed, overexpression of both LHCX2&3 has been shown to rescue NPQ in a low-NPQ ecotype of P. tricornutum (Pt4) (Taddei et al., 2016).
The findings for P. tricornutum may not be directly transferable to other pennate diatoms, as for instance 11 LHCX genes were discovered in the genome of the sea ice diatom Fragilariopsis cylindrus, none of which could readily be related to the four LHCX genes in P. tricornutum (Mock et al., 2017). In addition, the F. cylindrus genome contains an LHCX gene that is related to the LHCX6 in Thalassiosira pseudonana, whereas a similar sequence is absent in the P. tricornutum genome. The T. pseudonana LHCX6 protein has been hypothesized to be associated with Dtx binding and as such play a direct role in excess energy dissipation via NPQs during acclimation to prolonged HL stress . This protein, furthermore, is downregulated in fluctuating light conditions (Grouneva et al., 2016), underscoring its potential role in more prolonged oversaturating light conditions.
Recent studies using immuno-blotting revealed the presence of several light-regulated LHCX-proteins in natural communities and isolates of the microphytobenthic diatom S. robusta, of which some differ in size from P. tricornutum homologs Blommaert et al., 2017). To date, however, nothing is known about the diversity, organization and transcriptional regulation of these LHCX proteins in truly benthic diatoms. In the present study, we therefore identified LHCX genes in the S. robusta draft genome and followed their transcriptional regulation during a day/night cycle and during exposure to HL conditions. In addition, we investigated the conservation of potential pH sensing amino-acid residues.

Culture Conditions
Seminavis robusta strain 85A was obtained from the diatom culture collection (BCCM/DCG) of the Belgian Coordinated Collection of Micro-organisms 1 , accession number (DCG 0105). Diatom cultures were grown at 20 • C in semi-continuous batch culture in 1.8 L glass Fernbach flasks (Schott) under a day/night rhythm of 16/8 h with a light intensity of 20 µmol photons m −2 s −1 . Cells were cultured in Provasoli's enriched f/2 seawater medium (Guillard, 1975) using Tropic Marin artificial sea salt (34.5 g L −1 ) enriched with NaHCO 3 (80 mg L −1 final concentration). Cultures were acclimated to these culturing conditions for at least 2 weeks.

Day/Night Cycle and Prolonged Darkness
After acclimation, S. robusta was grown in 650 mL culture flasks (Greiner bio-one) to monitor LHCX expression during a 16/8h day/night cycle and in parallel during an extended dark cycle (during which cultures were kept in the dark for 24 h). Three biological replicates were sampled independently at 0:00, the start of the daily dark period, at 6:00, 2 h before the start of the light period, at 15 min, 1 h and 4 h after the start of the day period, and at the end of the light period (24:00). Gene expression was compared to gene expression in the samples at 6:00 h.

High Light Exposure
High light exposure was identical to the conditions described in Blommaert et al. (2017) and imposed about 6-8 h after the onset of the daily light period. Cultures in exponential growth were concentrated to 10 mg L −1 Chl a (determined spectrophotometrically, Jeffrey and Humphrey, 1975) by centrifugation at 4,000 RCF for 5 min and were allowed to recover in growth conditions (20 • C, 20 µmol photons m −2 s −1 ) for 2 h. Immediately before the start of the experiment, NaHCO 3 (4 mM) was added from a 2 M stock to prevent carbon limitation 1 http://bccm.belspo.be/about-us/bccm-dcg during the experiment. Four 65 W white light energy-saving lamps (Lexman) were used to provide HL conditions (2,000 µmol photons m −2 s −1 ) as used by Lepetit et al. (2013). Cells were continuously stirred in a glass test tube to obtain a homogenous cell suspension. The glass test tube was continuously cooled in a custom-made glass cooler by a water bath at 20 • C. Three biological replicates were sampled immediately before the onset of 2,000 µmol photons m −2 s −1 and after 15, 30, and 60 min of HL. Gene expression in the treated samples was compared to the samples before HL (0 min).

RNA Extraction and cDNA Synthesis
Four milliliters of cell culture was sampled each time on 3 µm Versapor filters (PALL Corporation). The filter was washed with ice-cooled phosphate buffered saline (PBS) and immediately frozen in liquid nitrogen. Samples were stored at -80 • C before RNA extraction. RNA extraction was based on Le Bail et al. (2008). Frozen samples were immediately incubated in 500 µL extraction buffer (100 mM Tris-HCl pH 7.5, 2% CTAB, 1.5 M NaCl, 50 mM EDTA, and 10% β-mercapto-ethanol) and subsequently beaten with carbid beads for 30 min in a beadbeater at 30 Hz. One hundred microliters of 10% Chelex-100 was added before the samples were incubated for 15 min at 56 • C with occasional vortexing. One volume of chloroform:isoamyl alcohol (24:1, Vol/Vol) was subsequently added before shaking the samples for 25 min at 5 Hz. After centrifugation, the upper phase was transferred to a new tube and mixed with 0.3 volume of absolute ethanol to precipitate polysaccharides. One volume of chloroform was added and after centrifugation the upper phase was transferred to a fresh tube. RNA was precipitated overnight at −20 • C, by adding 0.25 volumes of 12M LiCl and 1% (of final volume) β-mercapto-ethanol. The next day, the RNA was pelleted, dried and washed with 70% ethanol. Residual DNA was eliminated with DNAse I (Turbo DNAse, Ambion) according to the manufacturer's instructions. Extraction was performed with 1 volume Phenol-Chloroform (1:1, Vol/Vol). After centrifugation the upper phase was transferred to a fresh tube, extracted with one volume of chloroform:isoamylic alcohol (24:1, Vol/Vol) and centrifuged again. The upper phase was precipitated with 0.3 M NaOAc (pH 5.5) and 100% ice-cold ethanol by incubating for 1 h at −80 • C. After the samples were centrifuged for 20 min at 4 • C, the supernatant was discarded, and the pellet washed with 70% ethanol. The pellet was finally resuspended in RNAsefree water. The samples were reverse transcribed using Bio-Rad iScript cDNA kit.
Identification of LHCX Genes in the S. robusta Draft Genome LHCX sequences from T. pseudonana, P. tricornutum, F. cylindrus and Pseudo-nitzschia multiseries (with kind permission of E. V. Armbrust) were obtained from the JGI database 2 . These LHCX sequences were used to build an amino-acid HMM (Hidden Markov Model) profile using HMMer (version 3.1b1; Mistry et al., 2013), which was used to search all genes (annotation version 1.0) predicted for the 1 | Gene ID, GenBank accession number, Name, calculated molecular weight (Mw) and primer specificity. Name colours match the clade colours used in Figure 1. Primer specificity was tested using FastPCR in silico PCR with default settings. Green shading represents an amplified PCR product, whereas orange represents the possibility of an amplified gene product, however, with one of both primers having a melting temperature 40 • C < t m < 50 • C when binding on the corresponding LHCX transcript. As most LHCX3 sequences are rather similar in the primer regions, the primer LHCX3 picks up multiple related transcripts. The LHCX6 primer was designed on the SrLHCX6 RNA trailer and denoted with an "*".
This unedited alignment (383 amino-acid positions) was used for phylogenetic tree construction using IQ-tree (version 1.5.5; Nguyen et al., 2015) under the best amino-acid substitution model selected by the build-in model-selection function (ModelFinder) (Kalyaanamoorthy et al., 2017) allowing for the following set of potential models: JTT, LG, WAG, Blosum62, VT and Dayhoff. The FreeRate model (Yang, 1995) was chosen to account for rate-heterogeneity across sites and empirical amino-acid frequencies were calculated from the data. 10,000 ultra-fast bootstrapping cycles (Minh et al., 2013) were performed to validate clustering. LHCF sequences were used as an outgroup to root the tree, based on Nymark et al. (2013). Table S1). Primer specificity was tested in silico with FastPCR (PrimerDigital). Single nucleotide polymorphisms (SNPs) between the whole genome sequenced strain (D6) and the strain used in the experiments (85A) were identified using in-house RNAseq data  using Integrative Genomics Viewer (IGV, Broad Institute) and did not affect primer specificity. CDKA1, V4 and V1 (Moeys et al., 2016) were used for normalization as these were most stably expressed (Qbase + software). Qbase + normalized data is shown in Supplementary Figure S3. Log 2 expression ratios were compared with REST2013 software. The RT-qPCR program contained the following steps: pre-incubation: 95 • C -5 min, amplification: 95 • C -10 s, 58 • C -10 s, 72 • C -20 s (40 cycles), melting curve: 95 • C -5 min, 65 • C -1 min, 97 • C.

LHCX Presence in the Genome of S. robusta
A HMMER search, with a profile based on annotated LHCX genes from T. pseudonana, P. tricornutum, F. cylindrus and P. multiseries, yielded 17 putative LHCX sequences in the draft S. robusta genome (SrLHCX), all with a calculated Mw of about 20 kDa (Table 1), with exception of SrLHCX6a and SrLHCX6b, the latter being truncated on the C-terminus and possibly being a pseudogene, see Supplementary Figure S1. The resulting FIGURE 1 | Phylogenetic tree of LHCX genes in Seminavis robusta (Sr, colored), based on a multiple alignment made using MAFFT L-INS-i and constructed using IQ-tree. Total of 10,000 ultra-fast bootstrap approximation iterations were run (values are shown at the nodes). LHCX/LHCSR sequences are shown for the diatoms Thalassiosira pseudonana (Tp), Thalassiosira oceanica (To), Cyclotella cryptica (Cc), Phaeodactylum tricornutum (Pt), Fragilariopsis cylindrus (Fc), Fistulifera solaris (Fs), Synedra acus (Sa), Pseudo-nitzschia multistriata (Pmst) and Pseudo-nitzschia multiseries (Pm, with kind permission of E. V. Armbrust), the green alga Chlamydomonas reinhardtii (Cr), the moss Physcomitrella patens (Pp) and the brown alga Ectocarpus siliculosus (Es). maximum-likelihood phylogenetic tree (Figure 1) shows two main well-supported clades, the uppermost diatom-specific clade comprising centric as well as pennate diatom sequences, but lacking sequences from the pennate model P. tricornutum and the araphid pennate Synedra acus. SrLHCX6a and b are found in a subcluster containing also TpLHCX6 and FcLHCX6.
The lower cluster contains all other SrLHCX proteins, all P. tricornutum proteins and proteins from the green alga Chlamydomonas reinhardtii, the moss P. patens and the brown algae E. siliculosus. Most SrLHCX proteins (apart from SrLHCX6a and b and LHCX2) cluster together with PtLHCX3 in one relatively well supported cluster. Within this cluster, three well-supported subclusters can be distinguished (grouping the SrLHCX1, three and four sequences). The seven Synedra acus LHCX proteins also cluster closely together and are related to SrLHCX1,3&4.
On a lower hierarchical level, the relatedness between both Pseudo-nitzchia species and F. cylindrus is evident, as is the case for both Thalassiosira species and C. cryptica.

LHCX Gene Expression
We studied LHCX expression in S. robusta during 24 h of a 16 h light (20 µmol photons m 2 s −1 )-8 h dark cycle (Figure 2A). Cultures kept in prolonged darkness were sampled in parallel ( Figure 2B). Note that for several primer sets multiple related transcripts can be amplified; the specificity of primer sets is given in Table 1. We will refer to the primer pair that amplifies multiple SrLHCX3 transcripts as "LHCX3." Gene expression levels were compared to expression levels in samples 2 h before the light period (time point 6:00 in Figures 2A,B) and samples taken during the light period, compared to samples taken in parallel during the extended darkness treatment ( Figure 2C). As in some replicates SrLHCX2 and SrLHCX4a transcripts were not detected at 0:00, data for this timepoint is not shown. Due to considerable variance between technical replicates of SrLHCX6 throughout the 24-h cycle, these data are not shown.
All investigated SrLHCX genes showed a significant upregulation 15 min after the dark/light transition compared to 2 h before light onset (Figure 2B), which was not the case for samples kept in prolonged darkness ( Figure 2B). For samples kept in prolonged darkness only SrLHCX1a,b and SrLHCX2 were significantly upregulated 1 h after the light period would have started, compared to 6:00. Transcript levels for all investigated SrLHCX genes were significantly higher in the cells sampled 15 and 60 min after the dark/light transition, compared to cells which were kept in prolonged darkness ( Figure 2C).
In addition, we studied the expression of SrLHCX genes in S. robusta in response to HL (Figure 3). All investigated SrLHCX genes were highly upregulated after 15, 30, and 60 min of HL, compared to the LL before HL exposure, except for LHCX3h at 60 min. SrLHCX4b and SrLHCX6 showed significantly higher expression at 30 min, compared to 15 min of HL, whereas SrLHCX2 declined significantly in expression between 15 and 30 min of HL. Between 30 and 60 min of HL, LHCX2, LHCX3, and LHCX3h declined significantly in expression.
We investigated the presence of three amino-acid residues which are known to function as sensors of the thylakoid lumen pH in the LHCSR3 in Chlamydomonas reinhardtii and which are indispensable for NPQ functioning (Ballottari et al., 2016). Two of these are also present in P. tricornutum LHCX1-3 sequences, but only one in LHCX4 (Figure 4). SrLHCX6 contains none of the protonatable residues in C. reinhardtii as is the case for LHCX6 in T. pseudonana. The same residues are conserved in all SrLHCX sequences as in PtLHCX1-3, with the exception of SrLHCX4a,b,c which lack the same residue as PtLHCX4. However, unlike the PtLHCX4 sequence, the SrLHCX4a-c sequences contain a glutamate residue (E, highlighted in yellow), which may have a protonatable function.

DISCUSSION
As LHCX proteins play a central role in the NPQ mechanism of planktonic diatoms (Bailleul et al., 2010;Lepetit et al., 2013Lepetit et al., , 2017Taddei et al., 2016) and light responsive LHCX-proteins have been observed in benthic diatom isolates and communities Blommaert et al., 2017), we investigated the presence of LHCX genes in the benthic diatom S. robusta and studied their transcriptional regulation during HL conditions and a darkness/LL transition.
We detected 17 LHCX genes in S. robusta, which is a high number compared to the model diatoms P. tricornutum (4) and T. pseudonana (5) but in the same range as in the psychrophilic sea ice diatom F. cylindrus (11) (Armbrust et al., 2004;Bowler et al., 2008;Mock et al., 2017), the brown alga E. siliculosus (13) and the haptophyte Emiliania huxleyi (15) (see Dittami et al., 2010 for an overview and a comprehensive phylogenetic tree). Even though a comparable number of LHCX genes was discovered in both S. robusta and F. cylindrus, LHCX diversification in both raphid diatoms seems to have occurred independently as LHCX genes of both species were found to belong to different clades, with most diversity in S. robusta being related to PtLHCX3. Interestingly, a diversification of LHCX genes related to PtLHCX3 seems to have occurred as well in the araphid diatom Synedra acus. There seems, however, no general FIGURE 3 | LHCX transcription levels during exposure to high light (2,000 µmol photons m −2 s −1 ). Expression ratios are log 2 transformed and indicated by the color chart. Values are averages of three independent biological replicates and relative to the respective initial values (LL). Significant changes at p < 0.05 (Pairwise Fixed Reallocation Randomization Test performed by REST2006) are indicated with an asterisk.
trend in the amount of LHCX genes in the genomes of centric and pennate diatoms.
Even though a certain degree of functional redundancy can be expected due to the high number of LHCX genes in S. robusta, transcription appears to be light regulated for all the studied genes and all investigated LHCX transcripts were strongly upregulated in HL conditions. However, the limited specificity of the LHCX3 primer combination makes it difficult to generalize about LHCX genes in this clade. Our results, as such, do not allow to conclude why S. robusta has such a high number of LHCX genes. The expansion of gene families seems to be a general feature of S. robusta and is not exclusive to the LHCX genes. The large genome size of S. robusta allows for duplication events which could in turn lead to adaptive evolution (Osuna-Cruz et al., 2020). A large set of LHCX genes could be required to cope with highly variable light conditions. However, P. tricornutum only possesses four LHCX genes, and these still enable the species to rapidly adjust to a highly fluctuating light climate (Lepetit et al., 2017). In addition, the ability of motile epipelic diatoms to rapidly migrate away from strong light conditions could minimize the need for strong physiological photoprotection (Serôdio et al., 2001;Barnett et al., 2015;Laviale et al., 2016;Blommaert et al., 2018) and hence also the need for so many functional genes in S. robusta. Lastly, the presence of many LHCX genes may allow FIGURE 4 | Amino-acid alignment of regions 1 and 2 of Chlamydomonas reinhardtii CrLHCSR3, P. tricornutum LHCX1-4, T. pseudonana LHCX6, and all LHCX sequences of S. robusta. The highlights in green represent conserved pH-sensing residues, whereas red highlights represent the absence of a conserved pH-sensing residue. Glutamate residues (E) in SrLHCX4a-c are highlighted in yellow.
to integrate various environmental signals and stresses to finely regulate LHCX content (Taddei et al., 2016;Buck et al., 2019).
LHCX transcripts are present during a LL day/night cycle in S. robusta. This is consistent with the presence of an LHCX protein in LL conditions, as reported by Blommaert et al. (2017). This protein, as PtLHCX1, could provide a basal NPQ capacity localized near the PSII core (Nymark et al., 2009;Bailleul et al., 2010;Lepetit et al., 2013Lepetit et al., , 2017Taddei et al., 2018). As all SrLHCX transcripts (except SrLHCX6 for which data are lacking) were induced upon a dark/light transition, we cannot rule out that multiple SrLHCX proteins fulfill a similar role as PtLHCX1. Linking transcriptional data and immuno-blotting, in this case, is not straightforward as the used antibody (anti-LHCSR3, Bonente et al., 2011) was not specifically designed to recognize diatom LHCX isoforms. In addition, the large number of LHCX genes of similar sizes (Table 1) and differences in actual and predicted LHCX/LHCSR protein size, because of posttranslational modifications (Bonente et al., 2011), complicate the comparison of both datasets as was also observed for the discrepancies in transcriptional and translational regulation of LHCXs in P. tricornutum and T. pseudonana Lepetit et al., 2017).
High light-induced transcription of LHCX genes as observed in S. robusta was reported for planktonic diatoms (Nymark et al., 2009;Lepetit et al., 2013;Taddei et al., 2018) and may enhance the basal NPQ level provided by the LHCX protein(s) already present in LL (Taddei