# Nonphotochemical quenching and beyond: multi-layered photoprotection shapes light-stress tolerance across seasonal niches in Arctic diatoms

Dany Croteau, Sébastien Guérin, Mireille Savoie, Natalie Donaher, Mohammad M. Amirian, Marcel Babin, Douglas A Campbell, Johann Lavaud

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Dany Croteau, Sébastien Guérin, Mireille Savoie, Natalie Donaher, Mohammad M. Amirian, et al. Nonphotochemical quenching and beyond: multi-layered photoprotection shapes light-stress tolerance across seasonal niches in Arctic diatoms. Frontiers in Photobiology, 2026, 3, pp.1745893. 10.3389/fphbi.2025.1745893. hal-05469149

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EDITED BY

Rajagopal Subramanyam,
University of Hyderabad, India

REVIEWED BY

Milan Szabo,
HUN-REN Biological Research Centre, Hungary
Ranay Mohan Yadav,
University of Hyderabad, India

*CORRESPONDENCE

Dany Croteau,
✉ dany.croteau.3@ulaval.ca

¹PRESENT ADDRESS

Dany Croteau,
Institute of Integrative Biology and Systems,
Laval University, Québec, QC, Canada,
Johann Lavaud,
UMR6539 LEMAR- Laboratory of Environmental
Marine Sciences, CNRS, University Brest,
Ifremer, IRD, Institut Européen de la Mer, Institut
Universitaire Européen de la Mer, Plouzané,
France

RECEIVED 13 November 2025

REVISED 16 December 2025

ACCEPTED 19 December 2025

PUBLISHED 21 January 2026

CITATION

Croteau D, Guérin S, Savoie M, Donaher N,
M. Amirian M, Babin M, Campbell DA and
Lavaud J (2026) Nonphotochemical quenching
and beyond: multi-layered photoprotection
shapes light-stress tolerance across seasonal
niches in Arctic diatoms.
*Front. Photobiol.* 3:1745893.
doi: 10.3389/fphbi.2025.1745893

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Nonphotochemical quenching
and beyond: multi-layered
photoprotection shapes
light-stress tolerance across
seasonal niches in Arctic diatoms

Dany Croteau¹,²*†, Sébastien Guérin¹,², Mireille Savoie³,
Natalie Donaher³, Mohammad M. Amirian⁴,⁵, Marcel Babin¹,²,
Douglas A. Campbell³ and Johann Lavaud¹,²†

¹Takuvik International Research Laboratory, Université Laval, Centre National de la Recherche, Québec, QC, Canada, ²Département de Biologie and Québec-Océan, Université Laval, Québec, QC, Canada, ³Biology Department, Mount Allison University, Sackville, NB, Canada, ⁴Department of Oceanography, Dalhousie University, Halifax, NS, Canada, ⁵Department of Mathematics and Statistics, Dalhousie University, Halifax, NS, Canada

Throughout their species seasonal succession, diatoms of the Arctic Ocean experience a radical habitat transformation, from surviving the dimly lit winter within sea-ice or in the water column, to rapid growth under increasing irradiances, forming massive spring blooms beneath melting ice and later in open waters. Therefore, their evolutionary path has been moulded by the opposing challenges of maximizing light capture part of the year while maintaining highly efficient photoprotection capacities to limit photodamage upon bursts of supra-optimal illumination. Two main photoprotection mechanisms exist in diatoms i) nonphotochemical quenching (NPQ) supported primarily by the xanthophyll cycle (XC) and stress-related LhcX antenna proteins and, ii) a rapid repair cycle of photosystem (PS) II core protein, PsbA, upon photodamage. Previous studies suggest that freezing temperatures slow protein turnover and favour photoprotection strategies that rely primarily upon XC-NPQ in polar taxa. We aim to revisit this hypothesis by dissecting the high-light response of five Arctic diatom species that dominate contrasting ecological niches: sea-ice, marginal ice-zone and open waters. We exposed each species to a high-light stress and subsequent recovery period under low light, with and without, inhibitors of XC-NPQ (dithiothreitol) or of plastid protein translation (lincomycin), blocking de novo replacement of PsbA. We confirmed the crucial role of XC-NPQ in protecting PSII but also report unexpected observations that challenge our current understanding of psychrophile species response to light stress. First, the impact of lincomycin on PSII photoinhibition was stronger than that of DTT, despite PsbA turnover being undetectable by immunoblots in most cases. Second, while our data support planktonic species showing better tolerance to high light than sympagic species, we found unsuspected diversity in photoprotection strategies. We hypothesize that these differences support a gradient from conservative strategies, possibly optimized for survival in the extreme sea-ice habitat of sympagic species, to productivity-oriented strategies in open water planktonic species dominating during the bloom period. In the transforming,
brighter, Arctic Ocean, the adaptedness of this community-wide strategy scheme
could be undermined, shaking up the historical dominance of certain diatom taxa.

KEYWORDS

Arctic Ocean, diatoms, lincomycin, nonphotochemical quenching (NPQ), photodamage and PSII repair cycle, photoprotection, seasonal succession, xanthophyll cycle (XC)

Introduction

In the Arctic Ocean, microalgal growth is constrained by
seasonal light availability characterized by extreme photoperiod
cycles interacting with the snow and sea-ice cover formation and
melt dynamics (Ardyna and Arrigo, 2020). This radical seasonal
habitat transformation leads to shifts in light intensity which can be
stressful to photosynthetic life. Nonetheless, microalgae, and
particularly diatoms (Pierella Karlusich et al., 2025), thrive in the
winter-to-spring transition, and exploit the changing environment
through a highly productive seasonal species succession (Croteau
et al., 2022) (Figure 1A). Typically, pennate diatoms overwhelmingly
dominate sea-ice (sympagic) communities, whereas a mixture of
pennate species (such as Fragilariopsis and Pseudo-nitzschia genera)
and centric species (notably the Chaetoceros and Thalassiosira

genera) are found in water beneath snow-covered ice (Booth
et al., 2002; Degerlund and Eilertsen, 2010; Poulin et al., 2011;
Lafond et al., 2019), together with other phytoplankters such as
Phaeocystis sp. (Blais et al., 2017). As melting progresses, light
penetrates deeper in the water column and most of the time,
annual productivity climaxes in massive blooms consisting
mostly of centric diatoms extending to the marginal ice-zone
(ice-covers ≈50% of water) (Payne et al., 2024). In open waters,
as nutrient concentrations decline and grazing pressure surges,
different specialization patterns are observed among centric
species, found either near the surface or in the deepening
chlorophyll (Chl) a maximum (Balzano et al., 2017; Croteau
et al., 2022). Therefore, contrasted photoadaptation and
photoprotection strategies, spanning a broad range of growth
light optima and maximal growth rates, are observed in Arctic

<figure><img src="image_3.png" /><figcaption>FIGURE 1</figcaption></figure>

(A) Schematic representation of the typical niche/habitats occupied by the five studied Arctic diatom species over their seasonal succession (see Croteau et al., 2022, for details) and the tested hypothesis that ice-related species rely more heavily on xanthophyll cycle (XC)-dependent nonphotochemical quenching (NPQ) than rapid turnover of photosystem (PS) II core protein PsbA — possibly more prevalent in planktonic species — for photoprotection under prolonged high-light stress. (B–D) Boxplots of species-specific protein allocation traits potentially impacting high-light stress tolerance: PsbA normalized to chlorophyll (Chl) *a* (*n* = 9, except *Fragilariopsis cylindrus*, *n* = 7; as two replicates fell below detection) (B), molar ratios of the large Rubisco sub-unit (RbcL)-to-PsbA (C), and the PSII repair protease FtsH-to-PsbA (*n* = 3 or 6 in *Thalassiosira gravida* and *Chaetoceros neogracilis*) (D). Different letters indicate statistically distinct groups based on Analysis of Variance (ANOVA) followed by Tukey's post-hoc test (see Supplementary Data S1 for full statistical outputs).
diatoms occupying different ecological niches (Croteau et al., 2021; Croteau et al., 2022). Importantly, all Arctic diatoms' show high plasticity enabling survival under both prolonged winter darkness (Kvernvik et al., 2018; Kennedy et al., 2019; Joli et al., 2024) and supra-optimal light conditions (Gilstad and Sakshaug, 1990; Leu et al., 2016; Kvernvik et al., 2020; Croteau et al., 2022; Guérin et al., 2024). For example, sympagic species like *Nitzschia frigida* start growing under infinitesimal light intensities (Hoppe et al., 2024) and persist late into the melt season when averaged daily irradiance at the ice-water interface can exceed 100 µmol photons m⁻² s⁻¹ (Alou-Font et al., 2013; Galindo et al., 2017). Likewise, *Thalassiosira* and *Chaetoceros* species start growing under the sea-ice cover but can experience illumination of up to 1,000 µmol photons m⁻² s⁻¹ in open waters (Croteau et al., 2022). Additionally, light fluctuations are caused by advective transport beneath snow-covered sea-ice with heterogenous optical properties (Tao et al., 2024), sudden mixing events induced by shelf break (Randelhoff et al., 2018) and wind driven mixing which is being amplified under climate change (Ardyna and Arrigo, 2020).

While increasing light transmission to the Arctic Ocean (through shrinking sea-ice cover) has supported greater diatom productivity in recent years (Lewis et al., 2020), thus possibly more biomass being transferred to upper trophic levels, high-light stress can also decrease their nutritive quality (Leu et al., 2010; Duncan et al., 2024). Light stress also induces photodamage to photosystem (PS) II, which can eventually lead to photoinhibition (Campbell and Tyystjärvi, 2012). Therefore, uneven capacities to overcome intensifying light stresses among Arctic taxa could impact future community composition and ecosystem functioning. Upon excessive light absorption, reduced electron acceptors downstream of PSII accumulate and decrease its photochemical efficiency. Thus, excited Chl *a* lifetime and the likelihood of generating toxic radicals increase, promoting photodamages to PSII by oxidising its core proteins, PsbA and PsbD (sometime referred to as D1 and D2, respectively) (Kale et al., 2017). Limiting these deleterious effects is uniquely challenging in polar environments as consumption of ATP and NADPH by the Calvin-Benson-Bassham cycle is severely slowed down by low temperature (Young et al., 2015). Two main photoprotection features allow diatoms to limit photodamage and photoinhibition.

Firstly, the deployment of mechanisms favouring dissipation of excess absorbed light energy by PSII as heat — at the expense of competing processes like photochemistry and fluorescence — collectively termed nonphotochemical quenching (NPQ) (Goss and Lepetit, 2015). In diatoms, the main NPQ component is related to a one-step xanthophyll cycle (XC), by which diadinoxanthin (DD) conversion to diatoxanthin (DT) is favoured under high light (Goss and Jakob, 2010). Accumulating DT interacting with stress-related proteins (Lhcx) in PSII behaves as a quencher enhancing heat dissipation (Buck et al., 2021; Croteau et al., 2025a). In darkness or under low light, the opposite DT conversion to DD is favoured, but NPQ relaxation time varies broadly between species and conditions. In temperate diatoms, the XC-dependent NPQ component typically relaxes in 15–20 min (Goss and Jakob, 2010). Strikingly, sustained XC-NPQ with relaxation prolonged for more than 24 h in darkness is observed in some Arctic species, especially ice-related low light specialists (Lacour et al., 2018; Croteau et al., 2021). Moreover,

Arctic diatoms can contain enormous quantities of xanthophylls (Guérin et al., 2022; Giossi et al., 2025) implying high xanthophylls concentrations in the thylakoid lipid membranes (Lepetit et al., 2010), which could provide further photoprotection through antioxidants (Dall'Osto et al., 2010) or membrane fluidity regulation functions (Bojko et al., 2019).

Secondly, photoinhibition is countered by the PSII repair cycle, in which the FtsH hexameric protease degrades damaged PsbA/PsbD allowing replacement by functional ones (Campbell and Tyystjärvi, 2012). Before repair, broken PSII act as non-photochemical quenchers, thus protecting neighbouring active PSII (through so-called “photoinhibition quenching” or qI (Nawrocki et al., 2021)). So far, trade-offs between these NPQ and PSII turnover have been little studied in polar diatoms. From a handful of studies (Kropuenske et al., 2009; Petrou et al., 2010; Galindo et al., 2017; Lacour et al., 2018), the emerging big picture is that freezing temperatures restrict the velocity of PSII repair, such that polar species are more reliant upon XC-NPQ for photoprotection. This conclusion is supported by similar observations in cold-adapted green algae (Pocock et al., 2007; Ni et al., 2017), *Phaeocystis antarctica* (Kropuenske et al., 2009) and the temperature-dependence of PSII-repair rate in the temperate *Thalassiosira pseudonana* (Wu et al., 2012). The contributions of XC-NPQ and qI to total NPQ are often distinguished on the basis of their characteristic relaxation times; on the order of tens of minutes for DD to DT conversion (Goss and Jakob, 2010; Blommaert et al., 2021), and hours for PSII repair in temperate diatoms, which can also be further described via PsbA quantification by immunoblots (Wu et al., 2011; Campbell and Tyystjärvi, 2012; Nawrocki et al., 2021). However, the extent of sustained XC-NPQ in Arctic diatoms (Lacour et al., 2018; Croteau et al., 2021), with kinetics overlapping PSII repair, compromises the power of this approach (see rationale in Material and methods).

Consequently, to understand how divergent photoadaptive strategies support diatoms ecological success in the Arctic, and in turn impact the rest of the trophic chain (Leu et al., 2016), it is not sufficient to study their growth under stable light conditions (Hegseth, 1989; Gilstad and Sakshaug, 1990; Croteau et al., 2022; Guérin et al., 2024). We must also investigate how they overcome bursts of supra-optimal light intensities. We tested the hypothesis that Arctic species are more reliant on XC-NPQ than upon rapid PSII repair to overcome acute light stress (Figure 1A). We used the same panel of five species, dominating in contrasting seasonal niches from sea-ice to open waters, for which we previously documented a low-to-higher light specialization gradient (Croteau et al., 2022), and distinct XC-NPQ behaviour (Croteau et al., 2021). This unprecedentedly large species panel and comprehensive experimental design, for a study on high-light stress in polar microalgae, allow us to position previous assumptions in an ecological context. As such, we hypothesized that if rapid PSII-repair cycle plays an important role in Arctic diatoms, it would most likely be observed in late succession planktonic species, showing physiological traits more like their temperate counterparts (Schiffrine et al., 2020; Kvernvik et al., 2021; Croteau et al., 2022). All strains were subjected to identical high-light stress, either without or with drugs inhibiting XC-NPQ (dithiothreitol) or plastid genome translation (lincomycin), thereby preventing PSII repair, for three treatments in total. We report that ice-related
species tend to be more susceptible to photoinhibition than
planktonic ones. Like previous studies on polar taxa, we did not
observe pronounced PsbA degradation with immunoblots (Petrou
et al., 2010; Ni et al., 2017). Although the contribution of PSII repair
remains unresolved, we unexpectedly report that in four out of five
Arctic diatoms, plastid genome translation contributes as much, if
not more, than XC-NPQ to limiting PSII photoinhibition. Our
discussion brings a fresh perspective on the unsuspected
complexity and multifaceted photoprotection strategies of Arctic
diatoms in the context of their extreme habitats and beyond the
frontline provided by XC-NPQ.

Materials and methods

Diatoms culturing and acclimation

The same five Arctic diatom strains typical of contrasting
seasonal light niches as in (Croteau et al., 2021) were used;
*Nitzschia frigida* (kindly provided by A. Juhl), *Fragilariopsis
cylindrus* (CCMP1102, Culture Collection of Marine
Phytoplankton), *Thalassiosira gravida* (RCC5318, Roscoff Culture
Collection), *Chaetoceros neogracilis* (RCC2278) and *Chaetoceros
gelidus* (RCC2046). Throughout the manuscript, we use the term
ice-related species to refer to *N. frigida*, *F. cylindrus* and *T. gravida*
altogether (Figure 1A), but exclude the two *Chaetoceros* species
mostly found in open waters. The reader is referred to (Croteau
et al., 2022) for a thorough description of each species ecological
niche. Cultures were grown in seawater (Baffin Bay 67.48 N;
63.79 W), sterilised by filtration (0.2 µm) and enriched with f/2
medium plus silicates. Culture triplicates of 600 mL were acclimated
under 50 µmol photons m⁻² s⁻¹ (measured with a US-SQS/L spherical
quantum sensor (Walz, Germany)) and maintained in exponential
growth by diluting with fresh medium every two-to-three day. Cell
numbers and diameter were measured using a Beckman Multisizer
4 Coulter Counter (Miami, FL, United States) except for *N. frigida*
which required microscopy counting (Utermöhl method). The Chl *a*
concentration (estimated with a 10AU fluorometer, Turner Designs,
San Jose, CA, United States), cell diameter and growth rate were
monitored every two-to-three day. Our previous studies showed that
using this common growth light intensity enables high XC-NPQ
capacity (Croteau et al., 2021) in all species, while remaining
reasonably close to their maximal growth rate (Croteau et al.,
2022). Continuous 24 h illumination was used to avoid potential
biases due to diurnal rhythmicity of NPQ (Guérin et al., 2024).
Cultures growth and all experiments were conducted in a 0 °C lab.

Experimental design and variable
fluorescence approach

A Water-PAM fluorometer (Walz) was used to monitor
minimal fluorescence, F₀ (open reaction centres), with a blue
modulated measuring light and maximal fluorescence, F<sub>M</sub>,
probed with the same measuring light while a saturating multiple
turnover flash (6,000 µmol photons m<sup>-2</sup> s<sup>-1</sup>, 600 ms) reduces all PSII
electron acceptors (closed reaction centres). To let enough time for
sustained XC-NPQ to relax and measure the true maximal dark-

adapted quantum yield of PSII, F<sub>V</sub>/F<sub>M</sub>, calculated as (F<sub>M</sub>-F<sub>0</sub>)/F<sub>M</sub>, we
incubated cultures of different species to varying time in darkness
which we had previously defined in (Croteau et al., 2021). These
dark incubation times ranged from 24 h for *N. frigida* and *F.
cylindrus*, to 12 h for *T. gravida*, and to 20 min for *C. neogracilis*
and *C. gelidus*. With NPQ fully relaxed, we also measured the PSII
functional absorption cross-section (σPSII) with a single-turnover
flash (10,000 µmol photons m<sup>-2</sup> s<sup>-1</sup>, 100 µs) of blue light (455 nm)
applied by a Fluorescence Induction and Relaxation (FIRe)
fluorometer (Satlantic, Canada, curves fitted with the MATLAB
software using the FIReWORX script written by Audrey Barnett
(https://sourceforge.net/projects/fireworx/)). Dark-acclimated
cultures were then transferred to a custom-made glass vessel
lightened with LED and maintained agitated with a magnetic
stirrer. A light stress was then applied by exposing the cultures
to 250 µmol photons m<sup>-2</sup> s<sup>-1</sup> for 2 h, an ecologically relevant common
light-stress level. Converted into daily irradiances considering the
chosen 24 h photoperiod, 250 µmol photons m<sup>-2</sup> s<sup>-1</sup> is comparable
with the irradiance sea-ice species experience during their last days
of growth, or Arctic planktonic cells at the deep chlorophyll
maximum being brought up to the surface by vertical mixing,
making it suitable for our comparative ecophysiological study
(see Figure 1 in (Croteau et al., 2022)). Crucially, the period of
sea-ice breakup corresponds to the broadest overlaps between each
species niche, while more intense light stress, combined with
nutrient scarcity, is a challenge predominantly faced by late-
season specialists like the *Chaetoceros* genus. The light-stress
treatment was then followed by a recovery period of at least 1 h
at 5 µmol photons m<sup>-2</sup> s<sup>-1</sup>, which was extended in the species for
which it was insufficient to fully relax NPQ in control treatment, i.e.,
the described light stress here without inhibitor addition (see
Results). The same experiment was repeated for three treatments
i) control, ii) with addition of dithiothreitol (DTT) (500 µM final
concentration (Lavaud et al., 2002)) to inhibit DD de-epoxidation to
DT and therefore XC-NPQ and, iii) with addition of lincomycin
(500 µg/mL final concentration (Wu et al., 2012)) to inhibit plastid
genome translation, thus PsbA *de novo* synthesis, and thereby PSII-
repair (Campbell and Tyystjärvi, 2012).

At different time points (see Results), 3 mL aliquots were
sampled and transferred into the Water-PAM cuvette and left in
the dark for 15 s before measuring minimal and maximal
fluorescence, termed F₀' and F_M' respectively. In this context,
F_V'/F_M' represents the potential quantum yield of PSII in the
dark, for a given NPQ state, rather than a true maximal (F_V/F_M)
or effective (ΔF/F_M' or φPSII) quantum yield, measured under
actinic light. We note that 15 s is possibly not long enough for
full reoxidation of PSII acceptors in the context of high-light stress
(which would increase the measured F₀'), therefore we limited our
analysis of variations in F_V'/F_M' to the subsequent recovery
period under low light (5 µmol photons m⁻² s⁻¹). We made this
compromise — using a short 15 s in the dark before measuring
variable fluorescence — to avoid underestimating NPQ due to the
potential relaxation of fast NPQ components. The quantum yield of
NPQ (φNPQ) was calculated as F₀'/F_M' - F₀'/F_M and NPQ as F_M'/F_M' -
1. Because some Arctic diatom species show sustained XC-NPQ
relaxation overlapping with times characteristic of PSII-repair, it is
not possible to distinguish XC-NPQ from qI simply by analysing
relaxation kinetics as in temperate organisms (Lazár, 2015). Instead,
we used the following logic. The $F'_V/F'_M$ levels are constrained by two factors: i) any process that compromises photochemistry (like photodamage) and, ii) kinetic competition with nonphotochemical quenchers. To disentangle these effects, we can predict how NPQ alone — assuming a Stern-Volmer relationship — should affect $F'_V/F'_M$. Deviations from this predicted value can then be attributed to loss of photochemistry due to photodamage more confidently. To do so, we used the photochemical quenching (qP), parameter as a diagnostic tool (with supporting evidence for the validity of this parameter in diatoms provided below). Originally introduced to estimate the extent of photochemical quenching in a sample exposed to light (Bradbury and Baker, 1984), qP is calculated as:

$$qP = \frac{F'_M - F_S}{F'_M - F'_0}$$

Where $F_S$ is the minimal fluorescence in steady-state under actinic light, and $F'_0$ is the minimal fluorescence expected if all reaction centres were open while NPQ remains active. The crucial $F'_0$ is often unavailable experimentally, but can be derived experimentally as (Oxborough and Baker, 1997):

$$\text{theo.}F'_0 = \frac{F_0}{\frac{F_V}{F_M} + \frac{F_0}{F'_M}}$$

if it is assumed that all decrease in maximal fluorescence ($F'_M$) is imputable to a Stern-Volmer-like quencher. In our case, with samples exposed to low light during recovery and then incubated 15 s in darkness, we have access to a measured $F'_0$ which can be compared to its theoretical value. Therefore, in:

$$qPd = \frac{F'_M - F'_0}{F'_M - \text{theo.}F'_0}$$

qPd gives variable fluorescence during low light relaxation, normalized to what it would be if the only process influencing $F'_M$ and $F'_0$ was a Stern-Volmer-type quenching (qPd = 1 under this condition). Values of 1-qPd above 0 (i.e., $F'_0$ > theo. $F'_0$) indicate the extent of PSII photochemistry limitation caused by persistent reduced electron acceptors or photodamaged reaction centres, independently of NPQ level (see in plants (Ruban and Murchie, 2012)). Many reasons suggest this approach to distinguish decrease in $F'_V/F'_M$ mediated by NPQ from photoinhibition could be even more powerful in diatoms than in plants, and allow circumvention of some of the difficulties that sustained XC-NPQ entails in Arctic species; i) the theoretical relationship between NPQ and PSII photochemistry has been validated in some pennate diatoms (Croteau et al., 2025a), ii) the Stern-Volmer linear relationship between NPQ and DT is robust in diatoms (including the five species used here (Croteau et al., 2021)) and, iii) diatoms lack state transitions (a non-Stern-Volmer-like photoprotection mechanism) (Owens, 1986). However, caution in data interpretation remains crucial as NPQ dynamics in these Arctic species have yet to be thoroughly examined. Moreover, during prolonged and intense light stress, changes in PSII cross-section, due to Chl a bleaching or de novo synthesis of PSII antenna/pigments for instance, are possible and would affect reference values of $F'_M$ and $F'_0$. We monitored pigment concentrations to confirm that Chl a remained roughly stable (Supplementary Figure S1).

## Pigment sampling and quantification

Before, and at various time points during high-light stress and following low light recovery, sub-samples of 15 mL were collected and quickly filtered onto GF/F filters (Whatman®, 0.7 µm, 25 mm), flash frozen in liquid nitrogen and stored at -80 °C until analysis. Samples extracted in 100% methanol were mixed (70:30, vol:vol) with a buffer solution (tetrabutylammonium acetate (28 mmol L⁻¹)) following a method adapted from Ras et al. (2008). Pigments content was measured using high-performance liquid chromatography with a Zorbax Eclipse XDB-C8 3.5 µm column (Agilent Technologies, Santa Clara, CA, United States). The de-epoxidation state (DES, in %) of the xanthophyll pool was calculated as:

$$DES = \frac{DT}{(DD + DT)} \times 100$$

## Protein extraction and immunoblots

Samples of 25–35 mL were collected for characterization of protein allocation traits (before high-light stress or inhibitor addition). The number of replicates was always at least three (triplicate in control treatment) and extended up to 9 for some species/protein targets. Additionally, at various time points during high light experiments material was sampled to monitor PsbA degradation/repair kinetics, for all treatments (except only control in *F. cylindrus*) and possible regulation of Lhcx isoforms, for only the control treatment. Samples were filtered onto GF/F filters, flash frozen in liquid nitrogen and stored at -80 °C until extraction. Filters were thawed and placed in FastPrep lysing vials with 400 uL of 1X Protein Extraction Buffer (PEB, Agrisera (Sweden) AS08_300). Cells were lysed for 3 cycles of 60 s at 6.5 m s⁻¹ with 60 s intervals on ice between cycles. The lysing vials were centrifuged at 16,100 x g for 5 min, the supernatant, which contained cell debris, was transferred to a 600 µL microcentrifuge tube and centrifuged again at 16,100 x g for 3 min. The resulting supernatant was transferred to another 600 uL microcentrifuge tube and stored at -80 °C until analysis. Immunoblots analysis was carried out as described in (Wu et al., 2012), except that the FCP6 blot was incubated overnight with a 1:5,000 dilution of the primary antibody in 2% ECL blocking solution immediately after transfer. Primary antibodies purchased from Agrisera were diluted as follows: PsbA/D1 (AS05 084) 1:10,000, RbcL (the large sub-unit of Rubisco) 1: 10,000 (AS01 017), FtsH 1:5,000 (AS11 1789). The FCP6 antibody (provided by Dr. Erhard Rhiel) was diluted 1: 5,000. The Anti-FCP6 detects all four Lhcx isoforms in *Phaeodactylum tricornutum* (Lepetit et al., 2013), and samples of this species filtered after a similar 2 h high-light stress (1,000 µmol photons m⁻² s⁻¹) were used as pseudo-standards for this assay. After washing, all blots were incubated in a 1: 15,000 dilution of secondary antibody (anti-rabbit HRP, Bio-Rad, lot 64230791) and agitated gently on a rotator table for 1 h at room temperature. Chemiluminescence was detected using Amersham™ ECL Select (GE Healthcare Life Sciences, now Cytiva, RPN2235) and imaged with a VersaDoc CCD system
(Bio-Rad). Band densities were quantified using ImageLab software (v4.0, Bio-Rad) based on a standard curve.

## Curve fitting and statistical analysis

All curve fitting and statistical analyses were performed in R software version 4.3.0. The nls. multstart package (Padfield et al., 2021) was used to test different non-linear functions to fit φNPQ relaxation as a function of low light recovery time (t) and a Hill function was selected based on the lowest median Akaike Information Criterion across species × Treatment (more details in Supplementary Figure S2):

$$ \Phi_{NPQ} = \Phi_{NPQ_{M}} - \left( \Phi_{NPQ_{M}} \times \left( \frac{t^{n}}{t^{n} + t_{50}^{n}} \right) \right) $$

where φNPQ_M is the maximum extrapolated φNPQ value immediately at light extinction, t50 is the recovery time for which half of φNPQ has relaxed and n is a sigmoidity factor.

We integrated this analysis in a comprehensive framework including previous studies on this species panel (Croteau et al., 2021; Croteau et al., 2022), to scrutinize how different photophysiological parameters may shape interspecies high-light stress response on statistical basis. We focused on parameters that reflect the interplay of adaptation and acclimation before exposure to high-light stress, encompassing growth rate, PSII integrity and light absorption, productivity assessed through ¹⁴C-uptake curves and PSII electron transport, photoprotection provided by NPQ and the XC, and protein allocation strategies (total of 19 parameters). Because not all parameters were measured simultaneously on the same biological replicates, and we aimed at capturing variation arising from adaptation and acclimation rather than replicate-level noise, we used the mean values from three independent replicates for each species. Analysis of Variance (ANOVA) followed by Tukey post-hoc tests were used to confirm significant differences between species for a given parameter.

A Principal Component Analysis (PCA) on the 19 acclimation parameters across the 5 species (mean centred and scaled) was conducted to reduce dimensionality and visualize the top contributors in a biplot. We previously observed that during low light recovery, $F_V'/F_M'$ and qPd behave differently across species, thus providing complementary information (see Results). Therefore, we summed the value of both parameters after 5 and 60 min of low light recovery and divided them by four (giving a value bounded by 0 and 1) to obtain a comprehensive index of PSII inhibition for all species and treatments. We appended these computed PSII inhibition indices to their associated species × treatments, resulting in a total of 22 parameters. We then calculated Pearson correlation coefficients among the parameters using cor() function in R.

# Results

## Protein allocation strategies

All protein allocation traits explored revealed significant differences between species (ANOVA and Tukey's post-hoc test,

P-value <0.05) (Figures 1B-D). The molar ratio between the PsbA PSII core protein and Chl a was the highest in the open water *Chaetoceros gelidus* (0.69 ± 0.26 mmol·mol⁻¹) and the lowest in the dual sympagic/planktonic life form *Fragilariopsis cylindrus* (0.15 ± 0.07 mmol·mol⁻¹) (Figure 1B). *Nitzschia frigida*, *Thalassiosira gravida* and *C. neogracilis* showed intermediate values (between 0.25 and 0.41 mmol·mol⁻¹) significantly lower than in *C. gelidus*. The molar ratio between the large subunit of Rubisco and PsbA (a proxy of carbon fixation versus photochemistry capacities (Li and Campbell, 2017)) was significantly higher in *C. neogracilis* (222.91 ± 90.5 mmol·mol⁻¹) than in all species (as low as 21.14 mmol·mol⁻¹ in *C. gelidus*) except *F. cylindrus* (108.67 ± 36.83 mmol·mol⁻¹) (Figure 1C). A similar interspecific pattern was observed for the FtsH/PsbA molar ratio (a proxy for PSII turnover capacities (Li et al., 2016; Bonisteel et al., 2018)). The highest average values were observed in *C. neogracilis* and *F. cylindrus*, intermediates ones in *N. frigida* and *T. gravida*, and values one order of magnitude lower in *C. gelidus* (Figure 1D).

## NPQ and xanthophyll cycle (XC) kinetics over high-light stress and low light recovery

Culture of the five species were submitted to high-light stress (2 h under 250 µmol photons m⁻² s⁻¹) in three different treatments; control, with addition of DTT (inhibiting diatoxanthin (DT) conversion to diadinoxanthin (DD)) and with addition of lincomycin (inhibiting plastid protein translation, and therefore *de novo* PsbA synthesis). We first focus on NPQ dynamics during the high-light stress. All species reached substantial φNPQ; *C. gelidus* reached the highest values, close to 0.8, while *T. gravida* displayed the lowest values, especially in the first hour of stress (0.4–0.5 φNPQ) (Figure 2A). Under DTT treatment, φNPQ was, unsurprisingly, much lower, remaining ≤ 0.2 in all species during the first 15 min (Figure 2B). Subsequently, φNPQ increased roughly linearly with time in all species despite DTT addition, with the steepest increase in *N. frigida*. Comparing lincomycin to control treatments, *T. gravida* was the only species showing a noticeable increase in φNPQ, reaching a final value of 0.6 (lincomycin) compared to 0.5 (control) (Figure 2C).

During low light recovery (5 µmol photons m⁻² s⁻¹), *N. frigida* and *T. gravida* consistently showed slower φNPQ relaxation (Figures 2D-F, see also Supplementary Figure S2). Since φNPQ above 0.2 remained after 1 h of recovery in control treatment only for these species, we decided to keep monitoring φNPQ relaxation over a total of 6 h (*N. frigida*) and 12 h (*T. gravida*). *Chaetoceros gelidus* showed an outstandingly fast φNPQ relaxation half-time ($t_{1/2}$) of ≈1.5 min under control and lincomycin treatments (Supplementary Figure S2). Excluding DTT treatments, this relaxation half-time is approximately one order of magnitude faster than the second fastest $t_{1/2}$ measured, in *C. neogracilis* under lincomycin treatments ($t_{1/2}$ not available for control treatment in this species). Despite slower φNPQ relaxation in the three ice-related species, near full relaxation was reached at their respective last measurement time point (Figure 2). Under DTT treatment, the starkest contrast in the reversibility of φNPQ was seen in *C. gelidus*, with φNPQ remaining at roughly 0.25 after 1 h of low light recovery. Stable Chl a concentrations over the treatments
<figure><img src="image_4.png" /><figcaption>**FIGURE 2**
Kinetics of the quantum yield of nonphotochemical quenching (φNPQ) induction monitored over 2 h of high-light stress (250 µmol photons m⁻² s⁻¹) under control (A), with addition of dithiothreitol (DTT) (B) and with addition of lincomycin (C) treatments in all five Arctic diatom species investigated. Kinetics of φNPQ relaxation as a function of time over low light recovery (5 µmol photons m⁻² s⁻¹) under control (D), DTT (E) and lincomycin (F) treatments. Relaxation was tracked for at least 60 min (time values are log-transformed on the x-axis during recovery, log (60) is marked by a dashed vertical line) and extended until near-complete φNPQ relaxation in *Nitzschia frigida* and *Thalassiosira gravida* (up to 720 min, marked by a solid vertical line). φNPQ as a function of recovery time is fitted with a Hill function over the mean value of biological triplicates represented by the dashed coloured lines; fitted parameters and statistic outputs are found in *Supplementary Figure S2*.</figcaption></figure>

suggest persistent NPQ is not an artefact due to Chl *a* bleaching (Supplementary Figure S1). *Chaetoceros gelidus* also contrasted with the ice-related species in that its φNPQ relaxation was not slowed down by lincomycin addition (versus control treatment) (Supplementary Figure S2). However, despite slower φNPQ relaxation upon lincomycin addition for most species, no species showed a marked increase in non-reversible φNPQ between control and lincomycin treatments at the end of low light recovery.

Under control treatment, all species converted large amount of diadinoxanthin (DD) to diatoxanthin (DT) (Figure 3, the de-epoxidation state (DES) is shown in Supplementary Figure S3) via the XC. As expected in diatoms (Croteau et al., 2021), DT conversion strongly correlated with NPQ induction, with or without lincomycin addition (Supplementary Figure S4). The largest DT concentrations were reached in *F. cylindrus* (≈25 mol · 100 mol Chl *a*⁻¹), about twice as much as the second largest, *C. neogracilis*, and five times more than in *T. gravida* (lowest concentrations). *Fragilariopsis cylindrus* also reached the largest DES with 75%,

but the gap in DES compared to other species was narrower than for DT accumulation (all reached at least 60%) (Supplementary Figure S3). Treatment with DTT nearly fully abolished DT conversion in all species but *N. frigida* (Figure 3A), the species that also reached the highest φNPQ under DTT treatment (Figure 2B). In *N. frigida* and *F. cylindrus*, substantial DT remained after φNPQ had fully reversed (except with DTT) (Figure 3).

## Quantum yield of PSII and photochemical quenching over low light recovery

In Arctic diatoms, XC-NPQ relaxation kinetically overlaps with typical hour-scale range of PSII repair usually associated to the reversal of the slow, qI, component of NPQ (Wu et al., 2011; Lazár, 2015; Nawrocki et al., 2021). Indeed, in our previous study, the three ice-related species investigated here took more than 12 h to fully
<figure><img src="image_5.png" /><figcaption>**FIGURE 3**
(A–E) Kinetics of diatoxanthin (DT) concentration per chlorophyll (Chl) a molecules over 2 h high-light stress (250 µmol photons m⁻² s⁻¹, yellow bar) and over subsequent low light recovery (5 µmol photons m⁻² s⁻¹, gray bar), under control (white), dithiothreitol (DTT, red) and lincomycin (Linc., purple) treatments in the five Arctic diatom species investigated. Recovery was tracked for at least 60 min and extended until near-complete NPQ relaxation in *Nitzschia frigida* (A) and *Thalassiosira gravida* (B), for which time values are log-transformed minutes on the x-axis, log (60) is marked by a dashed vertical line, and 720 min is marked by a solid vertical line. A similar representation of the de-epoxidation state as a function of time is found in *Supplementary Figure S3* and DT relationships to NPQ are found in *Supplementary Figure S4*.</figcaption></figure>

relax NPQ when transferred from the growth light level used here to
complete darkness (Croteau et al., 2021). As an alternative, we here
chose to investigate the parallel dynamics of the maximal potential
quantum yield of PSII ($F_V'/F_M'$) and the potential photochemical
quenching parameter in the dark (qPd) during low light recovery.
The maximal potential capacity to use light for PSII photochemistry
during recovery is shown by $F_V'/F_M'$, but it does not discriminate
between losses due to a Stern-Volmer-like NPQ, damaged PSII or
persistently reduced PSII acceptors (closed reaction centres, not
expected in darkness). After 60 min of low light in control
treatments, the recovery of $F_V'/F_M'$ (relative to its initial value)
was almost complete in *C. neogracilis*, *F. cylindrus* and *C. gelidus*, but
below 70% in *T. gravida* and *N. frigida*, for which full recovery was
only reached after more than 3 h of low light (Figure 4A). Under
DTT treatment, $F_V'/F_M'$ recovery was clearly slower and did not
return to value as high as for control treatment, with a much
stronger effect on *N. frigida*, *T. gravida* and *C. gelidus*
(Figure 4B). With lincomycin addition, $F_V'/F_M'$ recovered value
after 60 min of low light was even lower than with DTT treatments
in all species but *C. gelidus* (Figure 4C). Interestingly, $F_V'/F_M'$
recovery seemed to plateau with lincomycin (more visible in *N.
frigida* and both *Chaetoceros*), which was not the case with DTT
addition for which recovery progressed nearly linearly with time for
the full duration of low light exposure.

With some caveats (see Material and methods), the potential
photochemical quenching in the dark, i.e., qPd, can be used to assess
decreases in PSII photochemistry beyond the effect of NPQ strictly
arising from a Stern-Volmer-like mechanism (see Supplementary
Figure S5 and (Ruban and Murchie, 2012; Croteau et al., 2025a)).
Accordingly, the reversal of processes that compromise PSII
photochemistry, like an unusually slow “reopening” of reaction
centres or the repair of damaged PSII, can be visualized by
plotting 1-qPd as a function of time during low light recovery. In
this representation, 1-qPd = 1 indicates complete PSII inactivation.
For example, after 5 min of low light recovery under control
treatment, 1-qPd equals roughly 0 in C. neogracilis (Figure 5A,
green symbols). This suggests that the ≈50% decrease in $F_V'/F_M'$
measured simultaneously in this species/treatment (Figure 4A) was
entirely due to XC-NPQ outcompeting PSII photochemistry, rather
than photodamaged PSII. To summarize these relationships, $F_V'/F_M'$
(normalized to the initial $F_V/F_M$ value) is plotted against 1-qPd in
Figure 6. A theoretical linear relationship with a slope of -1 is
expected if $F_V'/F_M'$ decreases solely through loss of
photochemistry capacities — either via photodamage or
reaction centre closure — rather than kinetic competition with
other quenchers (i.e., NPQ) (see Supplementary Figure S5 for
more details). As such, Figure 6A confirms that XC-NPQ strongly
influences $F_V'/F_M'$, but not necessarily qPd in the control
<figure><img src="image_6.png" /></figure>

**FIGURE 4**
Kinetics of recovery under low light (5 µmol photons m⁻² s⁻¹) of the dark adapted maximal potential quantum yield of photosystem II (F<sub>V</sub>'/F<sub>M</sub>'), normalized to its pre-stress value (F<sub>V</sub>/F<sub>M</sub>), under control (A), dithiothreitol (DTT) (B) and lincomycin (C) treatments in all five Arctic diatom species investigated. Recovery was tracked for at least 60 min (time is log-transformed on the x-axis, 60 min is marked by a dashed vertical line) and extended until near-complete NPQ relaxation (see Figure 2) in *Nitzschia frigida* and *Thalassiosira gravida* (up to 720 min, marked by a solid vertical line).

<figure><img src="image_7.png" /></figure>

**FIGURE 5**
Kinetics of recovery under low light (5 µmol photons m⁻² s⁻¹) of 1-qPd following high-light stress, under control (A), dithiothreitol (DTT) (B) and lincomycin (C) treatments in all five species investigated. 1-qPd, the potential photochemical quenching in the dark, serves as a proxy of photosystem (PS) II photochemical efficiency corrected for the effect of a Stern-Volmer nonphotochemical quenching (1-qPd=0 indicates that all PSII are unable to perform charge separation, see Material and methods). Recovery was tracked for at least 60 min (time is log-transformed on the x-axis, 60 min is marked by a dashed vertical line) and extended until near-complete NPQ relaxation (see Figure 2) in *Nitzschia frigida* and *Thalassiosira gravida* (up to 720 min, marked by a solid vertical line).

treatment. The exception is *C. gelidus*, for which the near-instantaneous ΦNPQ relaxation was not accompanied by complete F<sub>V</sub>'/F<sub>M</sub>' recovery (Figure 3), suggesting some PSII remained damaged or closed (1-qPd>0) (Figure 5) longer than it took XC-NPQ to relax (Figure 2). A *contrario*, under DTT treatment, decrease in F<sub>V</sub>'/F<sub>M</sub>' is strongly correlated to 1-qPd, suggesting damaged or closed reaction centres predominantly drive the decline in F<sub>V</sub>'/F<sub>M</sub>' rather than nonphotochemical processes competing with PSII photochemistry (Figure 6B). Additionally, the near agreement of these relationships with the theoretical model supports that there are no other type of PSII fluorescence losses involved, like state transitions or Chl *a* bleaching. Under lincomycin treatment, 1-qPd began relaxation at lower initial values than with DTT treatment, but its recovery was slower and stagnated at higher levels (Figure 5C). This suggests that some PSII cannot regain capacity for

photochemistry following high-light stress when plastid protein translation is inhibited. Overall, lincomycin treatments shows a situation intermediate to control and DTT, where both the XC-NPQ and a slowly reversible loss of PSII photochemical efficiency contribute to the decrease in F<sub>V</sub>'/F<sub>M</sub>' (Figure 6C).

Overall, analysing both F<sub>V</sub>'/F<sub>M</sub>' and 1-qPd allows to discriminate the influence of XC-NPQ — assumed to behave as a Stern-Volmer process — from that of other processes, including photodamages, on PSII photochemistry during low light recovery. The values of both F<sub>V</sub>'/F<sub>M</sub>' and 1-qPd at 5 and 60 min of low light recovery were averaged to derive a comprehensive PSII inhibition index — ranging from 0 to 1 in all species and treatments combinations — and used in the multivariate analysis.
<figure><img src="image_8.png" /><figcaption>**FIGURE 6**
The dark adapted maximal potential quantum yield of photosystem (PS) II ($F_v/F_M'$), normalized to its pre-stress value ($F_v/F_M$) (Figure 4), plotted as a function of 1-qPd (potential photochemical quenching in the dark (Figure 5)) for all data points measured during low light recovery following high-light stress under control (A), dithiothreitol (DTT) (B) and lincomycin (C) treatments in all five Arctic diatom species investigated. In the absence of processes competing with PSII photochemistry, such as xanthophyll cycle mediated nonphotochemical quenching (XC-NPQ) (inhibited in (B)), a direct relationship (dashed line) between norm. $F_v/F_M'$ and 1-qPd is expected. Increasing 1-qPd represents more PSII being compromised for photochemistry from photodamage (or persistent reaction centre closure). Over low light recovery, PSII repair can restore photochemistry (inhibited in (C)) and 1-qPd trends toward 0. See Supplementary Figure S5 for more details on the theoretical relationships between norm. $F_v/F_M'$, qPd and NPQ.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**FIGURE 7**
Linear regressions (forced y-intercepts to 1, dashed lines) of photosystem II reaction centre protein PsbA-to-chlorophyll (Chl) a ratios normalized to $t_0$ as a function of time during high-light stress (250 µmol photons m$^{-2}$ s$^{-1}$, yellow bar) and low light recovery (5 µmol photons m$^{-2}$ s$^{-1}$, gray bar) under control (white), dithiothreitol (DTT, red) and lincomycin (Linc., purple) treatments in two species dominating the extreme of the succession; sea-ice specialist *Nitzschia frigida* (A) and the open water planktonic species; *Chaetoceros gelidus* (B) are shown. When applying a Bonferroni correction to the P-value significance threshold (13 comparisons across the five species), the relationship for *C. gelidus* DTT treatment is the only one that was significant (P-value <0.004). Example blots for one biological replicate for each treatment of both species are shown in (C,D). Relationships for all species and treatment are found in Supplementary Figure S6, their linear regressions output in Supplementary Data S3, and unedited blots in Supplementary Figure S7.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**FIGURE 8**
Ratio to chlorophyll (Chl) a of Lhcx isoforms, detected with Anti-FCP6 (Lhcx1) antibody (same as in (Lepetit et al., 2013)), normalized to $t_0$ in *Nitzschia frigida* under control treatment. The dashed line represents a linear regression (forced y-intercept to 1) as a function of time during high-light stress (250 µmol photons m⁻² s⁻¹, yellow bar) and low light recovery (5 µmol photons m⁻² s⁻¹, gray bar) (A). An example blot in which samples of *Phaeodactylum tricornutum* (P.t.), post-exposure to a similar light stress (2 h, 1,000 µmol photons m⁻² s⁻¹), are loaded for different total protein concentrations is shown in (B). This blot is used as a pseudo-standard, knowing that anti-FCP6 detects all four Lhcx isoforms in *P. tricornutum*, a species for which Lhcx1, 2 and 3 can be upregulated under high light but, Lhcx1 is the only constitutive isoform (Lepetit et al., 2013). Anti-FCP6 did not work consistently for the other species. Unedited blots are found in Supplementary Figure S7.</figcaption></figure>

## Protein kinetics; PsbA turnover and Lhcx expression

To determine if some long-lasting NPQ (qI), or loss of PSII photochemistry capacity (qPd<1), could be linked to photodamaged PsbA proteins, we sampled material to quantify PsbA via immunoblots over the high-light stress and low light recovery kinetics (four time points spread between $t_0$ and after 1 h of recovery). Surprisingly, across all species and treatments, only *C. gelidus* under DTT treatment showed a negative correlation between PsbA/Chl *a* and time of experiment (*P*-value = 0.003, significant after Bonferroni correction for 13 relationships tested), with a modest slope leading to a ≈20% relative decrease in PsbA/Chl *a* at the end of low light recovery (Figure 7). This is in stark contrast with $F'_V/F'_M$ and 1-qPd often indicating 50%-to-75% loss of PSII photochemistry compared to pre-stress levels under both DTT and lincomycin treatments. We also attempted to detect increases in Lhcx isoforms accumulations (only for the control treatment) with a promiscuous Lhcx anti-body (FCP6) (Lepetit et al., 2013) originally developed for *Cyclotella cryptica* (Westermann and Rhiel, 2005). The anti-body worked consistently only for *N. frigida*, revealing a doubling in total Lhcx, distributed among at least two isoforms, after 60 min of low light recovery (Figure 8).

## Statistical analysis

We added the protein allocation traits (Figure 1) to a list of 16 photoacclimation parameters related to growth, PSII integrity, productivity assessed with ¹⁴C-uptake curves and PSII electron transport, and the characteristics of NPQ and the XC, determined in the same species and growth light intensities in previous studies (see (Croteau et al., 2021; Croteau et al., 2022) and parameters definition in Supplementary Figure S8). Interspecific differences in photoacclimation were largely explained by two components in a PCA space, explaining 50% and 30% of variance respectively (Figure 9A) (all PCA outputs in Supplementary Data S4). Parameters linked to the maximal rate of C-fixation (Croteau et al., 2022) were correlated with Rubisco/PsbA, and closely aligned with PC1, showing a clear distinction between *N. frigida*, *T. gravida* and *C. gelidus* on one extreme and *F. cylindrus* and *C. neogracilis* on the other. The biological meaning of PC2 was more amalgamated, with the main contributors including $F'_V/F'_M$, maximal φNPQ at one pole and the Chl *a* specific initial slope of C-fixation at the other (αChl, in Figure 9A). The longest distance in PC2 was between *T. gravida* and *C. gelidus* (Figure 9A). The Pearson correlation coefficients among these parameters were computed together with the PSII photoinhibition index (averaged $F'_V/F'_M$ and qPd at 5 and 60 min of recovery) under each treatment (Figure 9B) and the interspecific significant differences for all parameters are tested with an ANOVA followed by Tukey post-hoc tests (Supplementary Figure S8). This reveals that the maximal φNPQ is positively correlated with reduced PSII inhibition in control treatment ($r$ = 0.63), but this correlation is much stronger in lincomycin treatment ($r$ = 0.95) (Figure 9C). In DTT treatment, the inhibition of the main photoprotective mechanism, XC-NPQ, revealed strong correlations ($|r|$ > 0.7) between PSII inhibition and NPQ/DT, as well as the three protein allocation traits.

## Discussion

Considering the number of species included, the three treatments used, and the biochemical quantification of proteins and pigments in parallel to PSII fluorescence monitoring, this study represents one of the most comprehensive analyses of Arctic diatoms' (or any polar microalgae's) high-light response. Additionally, the five species investigated each occupy a distinct ecological niche over their seasonal succession, allowing us to link light stress tolerance to Arctic diatoms' unique ecology and with the support of our previous works on these same species (Croteau et al., 2021; Croteau et al., 2022). We targeted two key photoprotective mechanisms in diatoms: XC-related NPQ and PSII repair cycle. While confirming a central role for XC-NPQ in coping with high-light stress, our results additionally revealed unexpected patterns, challenging the usual assumption of a single dominant photoprotection mechanism and instead pointing to a more diverse set of strategies across Arctic diatom species succession.

The sympagic *N. frigida* and the marginal ice-zone planktonic, *T. gravida*, displayed heightened sensitivity to high-light stress, characterized by longer NPQ relaxation times (Figure 2), and severely compromised PSII photochemistry (Figures 4, 5). However, *F. cylindrus*, found in ice-covered waters and within
<figure><img src="image_11.png" /><figcaption>**FIGURE 9**
Principal Component Analysis (PCA) over the protein acclimation traits depicted in Figure 1, combined with 16 additional acclimation parameters retrieved from (Croteau et al., 2021; Croteau et al., 2022), definitions given in Supplementary Figure S8. The PCA shows the distribution of the five Arctic diatoms species in the space of its two main components, with the five parameters with the highest absolute contribution to either component shown (A) (EPC is "extra photosynthetic capacity" and αChl the "Chl a specific initial slope of C-fixation"). Heat map of pairwise Pearson correlations between photosystem (PS) II inhibition indices (from 0 to 1, where 0 means fully inhibited) for each treatment (control, dithiothreitol (DTT) and lincomycin (Linc.), see Material and methods) and the 19 above parameters with the process they relate to (complete definition of interspecific Analysis of Variance (ANOVA) for each parameter are shown in Supplementary Figure S8) (B). Strong positive correlations (r > 0.7) appear in dark blue, strong negative correlations (r < -0.7) in dark red, and near-zero correlations in white. Linear correlations between the PSII inhibition indices across treatments and five selected photoacclimation parameters (highlighted with black dots in (B)) are shown with their *r* values in (C); the maximum yield of nonphotochemical quenching (φNPQ<sub>M</sub>) and the slope of the NPQ to diatoxanthin relationship (NPQ/DT) (Croteau et al., 2021) (first two columns), and the protein allocation traits shown in Figure 1. The PCA outputs are provided in Supplementary Data S4.</figcaption></figure>

sea-ice brine channels, showed strong tolerance to light stress, comparable with the two open water *Chaetoceros* species under the control treatment. Using specific inhibitors to abolish each photoprotective mechanism in the following treatments, we surprisingly observed that the magnitude of the effect of lincomycin (blocking plastid protein translation and PSII repair) was either comparable to, or more severe than, DTT (blocking XC-

NPQ) in four out of the five species (see Supplementary Figure S8). This seriously undermines the prevailing notion that XC-NPQ is the dominant, and often solely considered, photoprotection mechanism at play in polar diatoms. Moreover, the only species which was clearly more affected by XC-NPQ inhibition than by lincomycin was *C. gelidus*, which is abundant at the end of the Arctic bloom season and in Atlantic influenced waters (Booth et al., 2002; Balzano et al.,
2017; Lafond et al., 2019). Interestingly, *C. gelidus* also tend to dominate at the subsurface Chl *a* maximum and below, which could promote a strategy reliant upon more nitrogen versus its congeneric *C. neogracilis* often found at the surface (see below) (Balzano et al., 2017). Nevertheless, these observations seem to negate our initial hypothesis that open water planktonic species, which evolved under slightly higher temperatures and tend to be more alike their temperate counterparts (Schiffrine et al., 2020; Kvernvik et al., 2021), would rely more on PSII repair cycle than do ice-related species (Figure 1A). Even more perplexing, we could not clearly associate impaired PSII photochemistry observed upon lincomycin addition to a decrease in PsbA as observed per immunoblots (Supplementary Figure S6).

Therefore, a parsimonious working model consistent with our dataset must explain, on the one hand, how *functional* PSII photoinhibition can be so pronounced while the loss of PsbA (assessed via biochemistry) is often small or undetectable, and on the other, why this process is strongly exacerbated when lincomycin inhibits the translation of plastid-encoded proteins. Although counterintuitive at first, there are precedents in earlier findings of fluorometric indicators of PSII *functional* photoinhibition (sustained NPQ-qI and lower $F_v'/F_M'$) not directly correlating with a loss of PsbA in polar taxa (Kropuenske et al., 2009; Petrou et al., 2010). We propose that delayed PsbA degradation (via FtsH), tightly coupled to *de novo* PsbA synthesis, may serve as an extremophile photoprotection strategy. In this scenario, damaged PSII reaction centres remain connected to the antenna and act as qI-type quenchers, distributing excitation pressure away from active centres (Nawrocki et al., 2021). Degradation of these centres would be delayed until new components are ready for immediate replacement, obscuring changes in PsbA abundance in immunoblots analysis. In this model, even if protein turnover is difficult to detect biochemically, PSII repair remains active, which is consistent with the exacerbated *functional* PSII inhibition upon lincomycin addition. However, this interpretation challenges previous studies (Kropuenske et al., 2009; Petrou et al., 2010; Lacour et al., 2018) which suggested that cold temperatures kinetically limit the contribution of protein turnover to the high-light response of polar taxa, a point discussed below. Delayed damaged PSII degradation promoting longer qI lifetime may complement the hallmark sustained XC-NPQ of Arctic diatoms (Lacour et al., 2020; Croteau et al., 2021) with a second slow-relaxing quenching mechanism originating from a different quencher. The slightly lower $F_v'/F_M'$ compared to what 1-qPd predicts in DTT treatment (Figure 6B) are consistent with such qI quenching being at play, whose effect becomes more apparent when XC-NPQ is inhibited.

Notably, even in temperate strains, inactivated PSII reaction
centres awaiting recycling can account for up to 60% of the total PSII
pool in diatoms (Campbell et al., 2013; Li et al., 2016). Another
surprising observation was that *C. gelidus* showed the highest PsbA/
Chl *a* ratio together with the largest functional PSII cross-section
(along with *F. cylindrus*) (Supplementary Figure S8). Chlorophyll *a*
being allocated to more PSII reaction centres should lead to lower
σPSII (assuming roughly equal Chl *a* allocation between PSI and
PSII among species), and both parameters were indeed negatively
correlated across the four other species (R² = 0.82) (Supplementary
Figure S9). This suggests that *C. gelidus* may maintain large pool of

"spare unassembled" PSII (Levitan et al., 2019) ready to replace damaged ones when needed, a photoprotection strategy also documented in the centric temperate *Skeletonema costatum* (Lavaud et al., 2016) and an Arctic *Micromonas* (Ni et al., 2017). In this case, degradation/replacement of inactivated PsbA can occur without needing *de novo* synthesis, which could explain why we measured a significant decrease in PsbA by immunoblots only in this species (Figure 7). Such a PSII repair strategy would also not be hampered, in the short term, by lincomycin treatment and may explain, in combination to its extremely high maximal φNPQ, the relative resistance of this species to this treatment (Supplementary Figure S8).

While the effect of lincomycin is often narrowly interpreted as
blocking PsbA turnover, it could also abolish regulatory strategies
relying on other plastid-encoded protein synthesis that are crucial
in Arctic diatoms' extreme environment. Before entertaining this
hypothesis further, we must ask if the timescale of our experiment,
combined with low temperature, kinetically allow for protein
translation to be a significant part of the strategy. Although
Lhcx proteins are nuclear-encoded, our results confirm that, at
least in *N. frigida*, rapid protein translation in response to high-
light stress is possible at 0 °C in an Arctic species (Figure 8). This
regulation at the protein level was is fast as in *P. tricornutum*
during a similar high-light stress conducted at 20 °C (Lepetit et al.,
2013). This Lhcx increase suggests that rapid PsbA turnover is at
least possible in Arctic diatoms, despite obvious methodological
challenges in monitoring it. Also supporting this proposition is
the fact that a significant decrease in PsbA was seen only in *C.
gelidus*, the species that showed the lowest FtsH content — which
typically correlates with lower proteolytic degradation rates
(Campbell et al., 2013).

Therefore, it is plausible that lincomycin's impact reflects not
only a direct constraint on the repair of PSII — i.e. reversing net
photoinhibition — but also indirect effects that favour the
occurrence of photodamage and gross photoinhibition (as
assessed by PSII fluorescence). Diatoms' plastid genome is
remarkably conserved and contains relatively few genes, most
of which encode subunits of major photosynthetic complexes (PSI
and PSII, cytochrome *b*<sub>6f</sub>, Rubisco, among others) (Yu et al.,
2018). Still, theoretically, the abundance of these complexes, or of
specific subunits, might be rapidly adjusted to help cope with
high-light stress. Crucially, the two potential bottlenecks to
photosynthesis in microalgae, plastoquinol oxidation by the
cytochrome *b*<sub>6f</sub> (Sukenik et al., 1987) or carboxylation by
Rubisco, are reliant on plastid-encoded subunits. Interestingly,
if the rate of plastoquinol oxidation can be accelerated via an
increase in cytochrome *b*<sub>6f</sub> stoichiometry, it could additionally
support enhanced capacity for cyclic electron flow (CEF), while
strong XC-NPQ favours the oxidation of the plastoquinone pool
(Croteau et al., 2025b). This alternative electron rerouting
produces extra ATP which may sustain PSII repair and could
relieve electron bottlenecks favouring PSII photodamages and
inhibition. In diatoms, the rate of CEF is often considered low
under non-stressful conditions, with extra-ATP production offset
by alternative electron fluxes towards the mitochondria (Bailleul
et al., 2015), whose operation has recently been reported in an
Arctic diatom (Rehder et al., 2025). Yet under stressful conditions,
typical of polar environments (Goldman et al., 2015) or during
high-light stress triggering large XC-NPQ (Croteau et al., 2025b), CEF is likely a crucial component of diatoms' regulatory toolkit. Likewise, an upregulation of Rubisco concentration is possible, which could increase the C-fixation rate and provide a larger energy sink on PSI acceptor side (see correlation between C-fixation capacities and PSII inhibition indices in Figure 8B). Crucially, polar diatoms compensate for slower catalytic rates at low temperature by accumulating large concentrations of Rubisco (Young et al., 2015), but a dynamic regulation of its abundance to overcome a sudden increase in light has yet to be documented. In all cases, if the inhibition of these putative processes by lincomycin contribute to its surprisingly pronounced impact on PSII photoinhibition, these hypotheses remain consistent with our dataset only when considered together with the delayed degradation of damaged PSII proposed above. We favour this model over alternative slow-relaxing quenching components, such as the qH mechanism described in higher plants (Malnoë, 2018), which we would not expect to be enhanced by lincomycin since its regulators are nuclear-encoded.

Given the lack of clear PsbA degradation patterns across taxa, we turned to a broader statistical approach to scrutinize other key factors potentially influencing Arctic diatoms' light stress resistance, combining the analysis of protein allocation traits shown in Figure 1 with our previous description of various photoacclimation parameters on the same species panel (Croteau et al., 2021; 2022). A PCA revealed that parameters related to maximal productivity explained the most variance among the five species and strongly correlated with PC1 (Figure 9A, plus all parameters contribution in Supplementary Data S4), for which *C. neogracilis* and *F. cylindrus* had values much higher than the three other species. Interestingly, PC1 was correlated with RbcL/PsbA, but also less intuitively, with FtsH/PsbA and lower slopes in the relationship between NPQ and DT. From coefficient correlations comparison (Figure 9B), and in view of the current state-of-knowledge, we propose that in our experimental design, three features favoured species' PSII tolerance to high-light stress — approximated by the PSII inhibition index computed in Results — when the bulk of photoprotection provided by XC-NPQ was abolished by DTT: i) The maximal rate of carbon fixation, ii) PSII repair cycle capacities (approximated by FtsH/PsbA) and, iii) an overlooked photoprotective role of xanthophylls free in the lipid membrane (Dall'Osto et al., 2010), as opposed to those PSII-bound and directly involved in NPQ (Lepetit et al., 2010; Croteau et al., 2025a); with higher free xanthophylls yielding lower NPQ/DT slopes and higher DD + DT pools (Figure 9C). Given the dominant role of XC-NPQ under control and lincomycin treatments, we could not clearly identify strong relationships between the PSII inhibition index and other photoacclimation features.

Nevertheless, these hypotheses remain based on a limited number of species and photoacclimation parameters analysed across our studies. Other important traits, for instance alternative electron flows (Goldman et al., 2015; Croteau et al., 2024; Rehder et al., 2025), or ROS production and detoxifying machinery (Schneider et al., 2016), could play differential roles in the high-light stress response among different species. Plus, biovolume, which has been shown to scale with some photophysiological parameters

(Finkel and Irwin, 2000; Key et al., 2010), also aligns particularly well with the high-light response scheme depicted here, especially under control treatment, from 15,000 µm³ in *T. gravida* to ≈40 µm³ in *F. cylindrus* and *C. neogracilis* (Table 1 in Croteau et al. (2021)). However, given the limited sample size spanning four orders of magnitude in biovolume, including it as a covariate seemed unadvisable. Finally, we must consider that the photoacclimation parameters used here represent acclimation to a single growth light intensity and do not reflect the full plasticity range of the species studied. For instance, while *F. cylindrus* displayed a high light tolerance similar to the open water *C. gelidus* here, we know from (Croteau et al., 2022) that its maximal carbon fixation rate and growth rate start to decline at lower growth light levels than *C. gelidus*. Eventually, achieving a complete depiction of how Arctic diatoms photoprotection capacities align with their seasonal niche, will also entail analysing how other environmental factors influencing physiology interact with light stress tolerance, such as temperature, pCO₂ and nutrient availability (Petrou et al., 2011; Schiffrine et al., 2020; Kvernvik et al., 2021; Rehder et al., 2024).

## Conclusion

Our findings confirm the crucial role of XC-NPQ in the high-light stress tolerance of Arctic diatoms, while also challenging the notion of a monolithic photoprotection strategy. We suggest an underappreciated role of xanthophylls not involved in NPQ (Dall'Osto et al., 2010; Bojko et al., 2019), and highlight the need to investigate for possible high-light-regulated putative proteins encoded in the plastid genome. As other before us, we failed to capture clear kinetics of PsbA degradation (Kropuenske et al., 2009; Petrou et al., 2010; Lacour et al., 2018). We propose that this reflects delayed proteolysis of damaged PsbA (Campbell et al., 2013; Li et al., 2016), such that degradation occurs only when it can be immediately followed by replacement via coupling with *de novo* synthesis. Overall, we show that high light tolerance among Arctic species aligns reasonably well with their seasonal niche, despite considerable interspecific heterogeneity in traits shaping photoacclimation strategies (Figure 9). Moreover, the marked specialization observed at both extremes of the succession could contribute to the outstanding ecological success of *N. frigida* and *C. gelidus*, which regularly dominate sea-ice communities (Poulin et al., 2011) and planktonic spring blooms (Booth et al., 2002; Lafond et al., 2019), respectively. *Chaetoceros gelidus* displayed uniquely fast XC-NPQ relaxation, favouring rapid return to maximal *potential* PSII efficiency such as in temperate counterparts (Lavaud et al., 2007), despite suffering from significant photodamages. This risky strategy may give *C. gelidus* the edge over competitors when racing for nutrients in bloom conditions. By contrast, the resilience-oriented strategy of *N. frigida*, characterized by sustained NPQ and the smallest PSII cross-section — making it a smaller photoinhibition target (Campbell and Tyystjärvi, 2012) — likely enhances survival in one of the most hostile environments for photosynthetic life. Global change rapidly disrupting the Arctic ecosystem (Ardyna and Arrigo, 2020) could lead to future misalignments between diatom species photoprotection strategies and their transforming niches, shaking up the prevalence of historically dominant taxa in this fragile environment.
# Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

# Author contributions

DC: Conceptualization, Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing. SG: Investigation, Methodology, Writing – review and editing. MS: Formal Analysis, Methodology, Validation, Writing – review and editing. ND: Formal Analysis, Methodology, Writing – review and editing. MMA: Formal Analysis, Methodology, Visualization, Writing – review and editing. MB: Conceptualization, Funding acquisition, Resources, Supervision, Writing – review and editing. DAC: Conceptualization, Formal Analysis, Methodology, Resources, Supervision, Writing – review and editing. JL: Conceptualization, Formal Analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Writing – review and editing.

# Funding

The author(s) declared that financial support was received for this work and/or its publication. We thank the contribution of CNRS in the framework of the IRL 3376 Takuvik, the Canada Excellence Research Chair on Remote sensing of Canada's new Arctic frontier (MB), NSERC Canada Discovery and Northern Supplement grants (RGPIN-2017-04505) (JL), the Sentinel North program of Université Laval (Canada First Research Excellence Fund) and the research network Québec-Océan for their financial support.

# Acknowledgements

We thank F. Bruyant for technical support, V. Richard for her support with HPLC analyses, Prof. A. Juhl and E. Riel for kindly

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# Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author JL declared that they were an editorial board member of Frontiers at the time of submission. This had no impact on the peer review process and the final decision.

# Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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# Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphbi.2025.1745893/full#supplementary-material

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