Romain Dupuis

Roland Pellenq

Alkali silica reaction: a view from the nanoscale

Understanding the formation of Alkali-silica reaction gel (ASR gel) and its behavior in cement pore network solutions is the key to mitigate one of the main causes of the strength retrogression of concrete. We herein propose a scenario to explain how ASR formation in cement pores can damage the cement paste structure that is supported by state-of-the-art atomistic simulations. We show the formation of an ASR gel at the atomic scale and how the drying or calcification affects its structure and strength. Moreover, we demonstrate that the alkali ions that are released during calcification are attracted by the gel and remain in its vicinity. The high concentration of ions in the solution modifies the interaction of the ASR gel with the cement pore walls and can lead to concrete damage.

I. INTRODUCTION

Cement is widely used as a construction material. It is a highly connected porous structure [START_REF] Bossa | Micro-and nano-x-ray computed-tomography: A step forward in the characterization of the pore network of a leached cement paste[END_REF] mainly composed of calcium silicate hydrate gel (C-S-H gel). After hardening, an important cause of cement loss of durability is the formation of alkali-silica reaction gels (ASR gel) in the pore network. The ASR gel formation is initiated by the dissolution of aggregates in mature cement pore solution but the reason why it creates cracks and damage in the cement matrix is not completely understood because of the complexity of the mechanisms. Over the recent decades, this phenomenon has motivated many studies [START_REF] Ramlochan | The effect of metakaolin on alkalisilica reaction in concrete[END_REF][START_REF] Bazantand | Mathematical model for kinetics of alkalisilica reaction in concrete[END_REF][START_REF] Thomas | Test methods for evaluating preventive measures for controlling expansion due to alkalisilica reaction in concrete[END_REF] showing the general need for improving the lifetime of cement and to reduce environmental costs [START_REF] Monteiro | Towards sustainable concrete[END_REF][START_REF] Jackson | Phillipsite and Al-tobermorite mineral cements produced through low-temperature water-rock reactions in Roman marine concrete[END_REF]. It is generally well accepted that ASR gel forms in the pore network due to the presence of a high concentration of alkali in a solution that stems from the dissolution of metastable minerals at high-pH. Nonetheless, there is no consensus on how this gel will create cracks in cement-based materials.

On one hand, studies of the ASR has lead to the general idea that ASR gel does not flow in the cement pore network due to its viscosity or its hardness [START_REF] Alnaggar | Lattice discrete particle modeling (ldpm) of alkali silica reaction (asr) deterioration of concrete structures[END_REF][START_REF] Baantand | Diffusion-controlled and creep-mitigated asr damage via microplane model. i: Mass concrete[END_REF][START_REF] Charpin | Modèle micromécanique pour l'étude de l'anisotropie de la réaction alcali-silice[END_REF][START_REF] Dunantand | Physically based models to study the alkalisilica reaction[END_REF][START_REF] Leemannand | E-modulus of the alkalisilica-reaction product determined by microindentation[END_REF][START_REF] Multon | Chemomechanical modeling for prediction of alkali silica reaction (asr) expansion[END_REF][START_REF] Ulm | Thermo-chemo-mechanics of asr expansion in concrete structures[END_REF][START_REF] Gholizadeh Vayghan | Compositionrheology relationships in alkalisilica reaction gels and the impact on the gel's deleterious behavior[END_REF][START_REF] Johnson | Fracture properties of the alkali silicate gel using microscopic scratch testing[END_REF]. In order to build up pressure that can damage the cement structure, the ASR gel must have to withstand more pressure than the cement pore walls. On the other hand, other studies suggested that ASR gel is a soft and compressible material, which can expand in the pore network of cement and which displays yield stress of 10 kPa [START_REF] Wu | Nano-scale structure and mechanical properties of asr products under saturated and dry conditions[END_REF][START_REF] Gholizadeh Vayghan | Compositionrheology relationships in alkalisilica reaction gels and the impact on the gel's deleterious behavior[END_REF] and a bulk modulus as low as 0.1 MPa [START_REF] Gholizadeh-Vayghanand | Quantifying the swelling properties of alkali-silica reaction (asr) gels as a function of their composition[END_REF]. It has been shown by electron spectroscopy measurements that the ASR gel is found in all the 2 microporous structures of the cement matrix, which indicates that it can flow in the pore network [START_REF] Wu | Nano-scale structure and mechanical properties of asr products under saturated and dry conditions[END_REF].

The disagreement between these different hypotheses stems from the variety and complexity of the systems as well as the difficulty in characterizing them. Moreover, ASR gel formation is followed by other mechanisms, such as alkali recycling due to the leaching of alkali ions from the ASR gel itself to the pore network solution. This could refuel the reaction of the ASR gel formation and a high concentration of alkali could also create dis-joining pressure if the ions remain in the vicinity of the gel [START_REF] Delville | Correlations between the stability of charged interfaces and ionic exchange capacity: a monte carlo study[END_REF].

A complete understanding of the atomistic structure is essential to predict how chemical conditions will influence the formation of ASR gel and its structure. So far, no atomistic models have been proposed to explain the formation and hardening of this gel, which is the key to understand the damage process. Herein, we propose an approach based on brief-parallel tempering [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF] (a method to accelerate the reaction of atoms) to observe the formation of the ASR gel in solution. We show that is a soft gel that can flow in the pore network. In that framework, we then simulate the several mechanisms that could transform the ASR gel: the gel drying over time due to water evaporation that will reinforce its mechanical properties; the calcification of the gel that will increase its viscosity and clog the pore network leading to damage due to the formation and growth of the ASR gel; and, the high concentration of ions in the pore solution at the interface between the ASR gel and the cement pore walls that will modify the interaction between the ASR and the C-S-H gels. Although these ions could play an important role in the pore network clogging and in building up pressure on the C-S-H pore walls, their localization at the atomic scale has not been thoughtfully studied yet.

II. SIMULATION METHODS

In order to determine the structure of ASR gels, we have first simulated the formation of such systems starting from a solution containing only silicate monomers, water and alkali cations (K + and Na + ). In Figure 1, a possible scenario of damage due to ASR gel formation is proposed. The gel formation corresponds to stage (ii) shown in figure 1, the gel starts to form in a solution containing highly dispersed silicates and alkali ions.

The formation of oligomers is a slow process compared to times reachable by Molecular 3 FIG. 1. Process of Alkali silica reaction gel formation leading to damage with ASR flow in the pore network. The C-S-H matrix is in grey, the water is in blue, the empty pore is in white, the ASR gel is in orange, the aggregates are in red and portlandite is in green. (i) Due to high pH, the formation of ASR gel is preceded by the dissolution of aggregates in the pore solution. ASR gel is formed in the solution in presence of an high concentration of silicates and ions. The ASR gel structure has to be determined from atomistic calculations. (ii) The soft gel grows in the highly-connected pore network due to the production of ASR gel. (iii) Calcification will harden the ASR gel and release ions into the pore network solution that will strengthen the ASR/C-S-H gel interaction.

The pore network will be clogged and the gel hardness is increased at the surface. The process of calcification is investigated herein. (iv) As shown hereafter, drying of the gel will participate to clogging the pore network if the pore network is not entirely saturated with water. Drying, which is still misunderstood at the atomic scale, can affect measurements of the mechanical properties.

Dynamics (MD) simulations due to the energy barrier of 0.2-0.6 eV to form Si-O bonds [START_REF] Raoand | Molecular dynamics simulations of the polymerization of aqueous silicic acid and analysis of the effects of concentration on silica polymorph distributions, growth mechanisms, and reaction kinetics[END_REF][START_REF] Dupuis | Doping as a Way To Protect Silicate Chains in Calcium Silicate Hydrates[END_REF]. We have used a recent accelerated simulation method, which is based on the parallel tempering method (PT), also called replica exchange [START_REF] Earland | Parallel tempering: Theory, applications, and new perspectives[END_REF]. In PT, the system of interest is replicated several times and each replica runs at different temperatures -high-temperature replica having a higher probability of crossing high-energy barriers. After a given time, two configurations can be exchanged. In the case of brief-PT, the PT is regularly interrupted leading to low-temperature states periodically repopulating the ensemble of simulations [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF].

The aim of such a method is to overcome large energy barriers that are limiting the range of times that can be reached by classical MD simulations. This enables us to observe the formation and dissociation of high-energy barriers and thus accelerate the dynamics by several orders of magnitude [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF][START_REF] Dupuis | Time resolved alkali silicate decondensation by sodium hydroxide solution[END_REF].

As described hereinafter, the gel that has been formed in those conditions is a non-viscous gel with relatively low connectivity. Therefore, we investigated a scenario in which the gel can flow in the pore network and can be transformed due to changes in the local chemical composition (drying or calcification).

The second stage of simulations consists in drying the system, which corresponds to stage (iv) shown in Fig. 1. To dry the system, we simply remove water molecules from the gel that has been previously formed and we monitor the temporal evolution of its microstructure.

The final stage of simulations corresponds to calcification [i.e., stage (iii) in Fig. 1]. The goal of this method is to study the replacement of Na + and K + in ASR gels by Ca 2+ and keep an interface between the gel and the surrounding solution. A standard molecular dynamics would not diffuse fast enough the cations to do the exchange since this process happens at longer time scales (i.e., in general months). For this, a new simulation cell is built. On the right side, it contains the hydrated gel that has been previously formed. On the left side it contains a solution containing Ca 2+ [ Fig. 4(a)]. The two parts are separated by a reflective wall that can not be crossed by ions. During the dynamics, we have made grand canonical Monte Carlo steps in order to substitute Ca 2+ with 2 Na + (or 1 K + and 1 Na + , or 2 K + ) in the solution. In this simulation, the ions from one side or the other of the wall are in interaction as if there was no wall. The purpose of this wall is to separate clearly the solution from the gel and avoid mixing both parts so as to study in detail the interface between the solution and the gel.

All the simulations were carried out with LAMMPS [START_REF]Triclinic (non-orthogonal) simulation boxes -lammps documentation[END_REF]. The system contains 100 Si atoms, 100 Na + and 10 K + (and 55 Ca 2+ ions for the calcification simulation). The whole simulation box contains 1100 water molecules and has the following dimensions 33x33x44 Å3 . Note that ASR gels composition in cement can vary from sample to sample [START_REF] Kawamuraand | Asr gel composition and expansive pressure in mortars under restraint[END_REF][START_REF] Gholizadeh-Vayghanand | The influence of alkalisilica reaction (asr) gel composition on its hydrophilic properties and free swelling in contact with water vapor[END_REF] and the alkali composition affects the gel's structure [START_REF] Balachandran | Characterization of alkali silica reaction gels using Raman spectroscopy[END_REF]. Herein, considering the computational cost of the method, we have simulated the gelation of a single alkali-silica gel with a model composition of Na/Si=1, Ca/Si=0.1, K/Si=0.1, and 40% water content. This configuration corresponds to experimental studies on ASR gel formation under controlled humidity [START_REF] Gholizadeh-Vayghanand | The influence of alkalisilica reaction (asr) gel composition on its hydrophilic properties and free swelling in contact with water vapor[END_REF].

The gels were synthesized under 95% relative humidity and contain approximately of 40% water for the gel composition that has been simulated herein.

The time step is 0.25 fs, in order to ensure that water molecule dynamics are correctly reproduced. Reactive potentials of the type ReaxFF [START_REF] Van Duin | Reaxff: A Reactive Force Field for Hydrocarbons[END_REF] have been employed, with the widely used parameter set of Forgaty et al. [START_REF] Fogarty | A reactive molecular dynamics simulation of the silica-water interface[END_REF]. Those parameters have been calibrated

to study silicate materials and reproduce the correct energy barrier for the formation of Si-O bonds compared to DFT calculations [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF], which is essential to study the formation of the ASR gels. It has shown good performances to reproduce the silicate gel formation [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF]. For the brief-PT method, we have used 16 replicas with temperatures ranging from 300K to 800K. The PT exchanges have been intended every 50 steps. ASR gel formation happens on a long time scale that is inaccessible with classical MD at room temperature, this method allows to quickly observe Si-O bond formation or dissociation that is necessary for the formation of the ASR gel, thus enhancing the dynamics.

A. Gelation

Nuclear magnetic resonance of 29 Si has long been used to study the structure of silicates.

By simulations, we can evaluate the quantity of Si in each site from Q 0 to Q 4 during the ASR gel formation. To catch the evolution of the speciation, we report in Fig. 2 the quantity Q n that reflects the connectivity of the silicate species in solution, where n is the number of Si-O-Si bonds associated to one Si (e.g. Q 0 is a silicate monomer). Since the simulation starts in a solution containing only silicate monomers, initially, all Si are in a Q 0 site. At the beginning of the simulation, the number of Q 1 rises from zero, while the first dimers are formed in solution. The quantity Q 1 quickly reaches a maximum of 50% of the total number of silicon atoms and we observe the increase of Q 2 , which indicates the formation of long linear chains of silicates. After convergence of the Q n distribution, almost half of the silicon atoms are in a Q 2 position. Moreover, the sum of Q 3 and Q 4 is about 45% leaving the remaining 5-10% for the Q 1 . This indicates that all the monomers have reacted and that the silicate chains that have been formed are long (as shown by the large quantity of Q 2 ) and contain rings, which is indicated by the presence of Q 3 and Q 4 .

show that most of the structures are dominates by Q 2 and Q 3 , which is what we observe in the simulation. In particular for Na/Si = 1, Q 2 are prominent in NMR and Raman spectroscopy measurements [START_REF] Balachandran | Characterization of alkali silica reaction gels using Raman spectroscopy[END_REF]. In recent studies, it has been shown that pure silicate gels have lower silicate connectivity than alkali-rich silicates [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF][START_REF] Dupuis | Time resolved alkali silicate decondensation by sodium hydroxide solution[END_REF]. In this work, we observe that ASR gels are more connected than pure silica gels simulated using the same method [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF][START_REF] Du | Atomistic origin of the passivation effect in hydrated silicate glasses[END_REF]. At the same water/Si ratio, the simulated ASR gel has Q

0 =0%, Q 1 =10%, Q 2 =40%, Q 3 =40%, Q 4 =10%, whereas the pure silica gel has Q 0 =10%, Q 1 =25%, Q 2 =40%, Q 3 =20%, Q 4 =5%
. This result indicates the role of ions in the structuration of silicate chains, i.e., the increase of crosslinks is solely due to the presence of ions in the initial solution. In the gel, ions have a dual role: they act as a charge compensator near a Si-O -groups that are more reactive than Si-OH groups, or they are solvated. Solvated ions will impede water molecules from hydrolyzing silicate chains and therefore help the formation of structures with more crosslinks [START_REF] Dupuis | Dissociation mechanisms of dissolved alkali silicates in sodium hydroxide[END_REF]. Note that Na + ions are also bond-makers in silica glass.

In order to compute the bulk modulus, we have used the elastic bath method [START_REF] Workumand | Improved simulation method for the calculation of the elastic constants of crystalline and amorphous systems using strain fluctuations[END_REF][START_REF] Carrier | Elastic Properties of Swelling Clay Particles at Finite Temperature upon Hydration[END_REF] developed for soft materials. In this technique, we couple the simulation cell to an elastic bath (here we have used a bath with a strength of 5 GPa). The calculated bulk modulus for the ASR gel is 0.1 MPa, which is in agreement with mechanical tests showing weak mechanical properties in humid ASR gels [START_REF] Rajabipour | Alkalisilica reaction: Current understanding of the reaction mechanisms and the knowledge gaps[END_REF]. This is one order of magnitude less than the C-S-H tensile strength of about 3 MPa [START_REF] Onuaguluchiand | Alkali silica reaction resistance of cementitious material containing cacl2-blended acrylic polymer emulsion[END_REF] and two to three orders of magnitude less than the bulk modulus calculated for C-S-H gel structures using ReaxFF [START_REF] Manzano | Elastic properties of the main species present in portland cement pastes[END_REF]. This value at 40% water content is in the order of magnitude of experimental values measured for the swelling pressured caused by ASR formation [START_REF] Gholizadeh-Vayghanand | Quantifying the swelling properties of alkali-silica reaction (asr) gels as a function of their composition[END_REF]. The simulation results show that the ASR gel, which is formed at high relative humidity in the cement pore network, is a nonviscous gel with low mechanical properties compared to the C-S-H gel. In order to create cracks in the cement structure, this gel has to overcome physical transformation in order to strengthen itself or to clog the pore network. Hereinafter, we investigate the role of drying and calcification on the structure of the ASR gel.

B. Drying

Drying has been identified as a possible cause of atomistic rearrangement in silica gels [START_REF] Wu | Nano-scale structure and mechanical properties of asr products under saturated and dry conditions[END_REF][START_REF] Vegaand | Study of structural evolution of silica gel using 1h and 29si NMR[END_REF]. One of the reasons is that dry gels could have a composition close to hydrated glasses and form siloxane bridges at low temperatures. This is likely to affect the results of experiments obtained in non-controlled humidity environments to measure the structural and mechanical properties of ASR gels [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF]. In order to investigate the effect of drying on the structural properties of ASR gels, we have removed half of the water molecule from the system (the water content is 20% of the mass in order to simulate water retention from the gel). The evolution of Q n , as shown in figure 2, is quick towards densification.

In Fig. 2(a andb), we observe on the right side that the drying also has an effect on the connectivity of the ASR gel. In particular, there is an increase in the number of Q 4 and a decrease in the number of Q 2 . However, the evolution of the connectivity is not as marked as it is for pure silica gels [START_REF] Dupuis | Molecular simulation of silica gels: Formation, dilution, and drying[END_REF]. This can be explained by the fact that ASR gel already contains 40% Q 3 and 5% Q 4 at 40% water content. Nonetheless, it is important to note that after drying, the silicate chains are percolating in two points (species that are connected all across the simulation cell). This is due to the densification of the gel at 20% water content (see Figure 4(b andd)). The percolation of gels at the atomic scale can be seen as the limit between a gel structure and a colloidal structure. This is reflected in an increase in the mechanical properties as we calculated that the dried gel has a bulk modulus of 10 GPa. This is consistent with the percolation which is observed in the system and it indicates the strength of silica gels is mostly due to the silica chains and the loss of durability of silicate structures is linked to the reverse oligomerization process [START_REF] Dupuis | Tracing Polymerization in Calcium Silicate Hydrates Using Si Isotopic Fractionation[END_REF][START_REF] Dupuis | Normal and anomalous self-healing mechanism of crystalline calcium silicate hydrates[END_REF]. This value is lower but in the same order of magnitude as the values reported for C-S-H gels in the literature (50-70GPa) [START_REF] Manzano | Elastic properties of the main species present in portland cement pastes[END_REF][START_REF] Pellenq | A realistic molecular model of cement hydrates[END_REF][START_REF] Moon | Mechanical properties of jennite: A theoretical and experimental study[END_REF]. Those values, calculated for the C-S-H gels, have been calculated on well-ordered atomic arrangements so the calculated values correspond to the highest expected values. More realistic values have been calculated recently using density functional theory calculations on amorphous C-S-H gel structures with defects. The reported values for Young's modulus range from 18 to 25 GPa [START_REF] Jia | Nano-scale modeling and elastic properties of a typical CSH (I) structure based on DFT and Molecular Dynamics Methods[END_REF], which is close to the value we have obtained for the dried ASR gel. Our results indicate that the bulk and Young's modulus of silica-based gels (e.g. N-A-S-H, C-A-S-H [START_REF] Puertas | A model for the C-A-S-H gel formed in alkali-activated slag cements[END_REF]) is strongly related to the connectivity in the gels and to the degree of percolation of the silicate chains. Ions, being in the solution, affect the mechanical properties indirectly by playing a bond-builder or bond-breaker role. Therefore, determining the atomistic structure of these gels and their connectivity is essential. In agreement with the experiments, we observe that the loss of water has a drastic effect on the structural properties of ASR gel [START_REF] Vegaand | Study of structural evolution of silica gel using 1h and 29si NMR[END_REF]. Such a drying could occur in different regions of the porous network and in particular in micro-cracks in which more crystalline ASR structures has been measured [START_REF] Wu | Nano-scale structure and mechanical properties of asr products under saturated and dry conditions[END_REF]. Note that in our simulations, the effect of drying is reversible if we increase the water content from 20% to 40%.

In summary, the drying does not have a large influence on the connectivity of the system but it drastically changes the shape of the silica chains and we observe percolation at HR=20%. These results indicate the importance of considering the humidity in the systems when doing measurements of the atomistic structure and mechanical properties of ASR gels.

Moreover, the drying could increase the viscosity and clog the pore network in the case of the formation of ASR gel in an unsaturated pore network [Fig. 1(iv)]. This could be one cause of the irreversible damages observed in cement structures. Other effects can also modify the atomistic structure and increase the mechanical strength, in particular calcification of the ASR gel.

C. Calcification

Calcification could reduce the flowability of the gel and add up with other mechanisms such as swelling (in particular due to water intake), clogging of the pore network, production of additional ASR gel, and lead to damage. It is important to understand the difference between divalent and monovalent ions in the structuration of the gels. The process of calcification has been simulated using the method described in section II and Fig. 1(iii).

Herein, we investigate the evolution of the silicate connectivity, the behavior of ions released in the solution.

Swelling of the ASR gel after calcification is often suggested as a cause of the cement damage because it could induce pressure on the cement pore walls [START_REF] Ichikawa | Alkalisilica reaction, pessimum effects and pozzolanic effect[END_REF]. As observed on Fig. 4(d,e), the calcification of the ASR gel shrinks the gel and increases its density. This suggests that in-situ, the swelling could be due to an intake of water. Experimentally, it has been observed that calcium in ASR gels modifies its swelling properties [START_REF] Gholizadeh-Vayghanand | The influence of alkalisilica reaction (asr) gel composition on its hydrophilic properties and free swelling in contact with water vapor[END_REF] and that some gels don't swell. In the simulations, the swelling can not explain pressure build-up on cement but the calcification will affect the atomistic structure in several different ways.

Therefore, it is necessary to investigate other effects than swelling that could be caused by calcification.

To separate the effects of drying and calcification, the calcification has been performed on the structure with 40% of water content. Figure 3 shows the evolution of the connectivity in the gel during and after calcification. The first result is that the number of crosslinks, indicated by Q 3 and Q 4 , has changed during the calcification. The number of Q 3 is reduced by about 10 and the number of Q 4 is increased by about 4. This is in agreement with experimental data [START_REF] Balachandran | Characterization of alkali silica reaction gels using Raman spectroscopy[END_REF] showing that gels with calcium have more Q 4 than gels with only alkali. The mean-chain length has increased and the longest silicate chain has percolated through the whole simulation cell (more than 33 Å). The final structure is mostly composed of Q 2 (50%) that corresponds to the bonding often observed in calcium silicate hydrate gels [START_REF] Rejmak | 29si nmr in cement: A theoretical study on calcium silicate hydrates[END_REF]. When the simulated gel calcifies, the bulk modulus of the gel increases by 2 orders of magnitude (from 0.1 GPa to 10 Gpa).

In the initial stage [Fig. 4(a) and (b)], the density of silicates is almost constant across the simulation cell -in the gel side of the simulation box. After exchanging calcium ions, we can observe a densification of the silicates towards the edge in contact with the solution [Fig. 4(d)]. The density distribution of Ca 2+ follows a similar trend since most of those ions have been exchanged with the solution. Meanwhile, the density of Na + near the reflective wall surface is higher than far from the surface.

D. Role of alkali ions during calcification

The released alkali ions remain in the vicinity of the ASR gel and do not diffuse freely in the gel's pore solution because they are interacting with the negatively charged ASR gel surface. Note that this effect is not observed with Ca 2+ ions before ionic exchange. This can be explained by the fact that in the gel, Na + and K + , being monovalent, will compensate the charge locally near the Si-O -sites whereas Ca 2+ , being divalent, will compensate the charge for two Si-O -sites. As a result, after calcification, the charges on the silicate chains are partially but not completely screened by Ca 2+ ions in the gel.

It is often suggested that alkali ions are "recycled" after calcification to dissolve aggregates and pursue the alkali-silicate reaction [START_REF] Figueira | Alkali-silica reaction in concrete: Mechanisms, mitigation and test methods[END_REF]. Herein we show that alkali ions that remain attracted by the ASR gel after leaching will not fully be reused to dissolve more aggregates as often suggested in the models. In our model, only 20% of the alkali ions are localized at a distance larger than 5 Å from the ASR gel surface. Note that these ions could diffuse at longer time scales in the solution and reused to form ASR gel.

The presence of alkali ions near the ASR gel surface will drastically change how the ASR gel interacts with the cement pore walls that are negatively charged at the surface due to the presence of anionic groups. For instance, the interaction between C-S-H nanoparticles in cement is mostly due to the presence of ions in the solution in-between the nanoparticles [START_REF] Gmira | Microscopic physical basis of the poromechanical behavior of cement-based materials[END_REF]. In line with primitive model results [START_REF] Delville | Correlations between the stability of charged interfaces and ionic exchange capacity: a monte carlo study[END_REF], the disjoining ASR-CSH osmotic pressure that develops could be due to this alkali layer at the interface between the rigid-CSH wall and the rigid Ca-ASR gel. Hence our results support that the origin of cement paste degradation due to ASR results from the Ca to Na exchange over time, which can happen over years. This paves the way to reconstruct the interaction potential between two ASR gel aggregates using a fully atomistic approach. Moreover, a study of the potential mean force acting on the C-S-H and on the ASR gel (using the microstructures) for different alkali concentrations could give interesting insights on the poremechanical properties of this system [START_REF] Ioannidou | The crucial effect of early-stage gelation on the mechanical properties of cement hydrates[END_REF][START_REF] Zhou | Multiscale poromechanics of wet cement paste[END_REF] and in particular on the influence of the ionic concentration and the type of ions (monovalent or divalent).

The fact that ions remain close to the ASR gel opens new areas of research to try to mitigate ASR damage at a low cost. To understand mitigation, the investigation of the role of ions appears to be key since they play a role at all stages of the ASR gel transformation (during the dissolution of aggregates, during the formation of ASR gel in the pore network, and during calcification). Since the ASR gel is mostly material with low mechanical properties compared to the C-S-H gel, understanding the diffusion of ions during calcification by atomistic simulation is essential. In order to mitigate ASR damage, one option would be to remove the alkali from the solution in the interlayer between the C-S-H and the ASR gels.

For this, the models that we propose can be deployed to study the competition between different ions and their affinity to the C-S-H and the ASR gel surfaces. For instance, lithium salts are used in ASR gel mitigation [START_REF] Islam | Comparison of asr mitigation methodologies[END_REF] but it is still unclear how it can mitigate damage.

Little is known on the role of lithium as it has primarily been considered as a mitigator of the aggregate dissolution, this could be assessed with the method developed herein.

More recently, studies have shown that it is possible to mitigate the freeze-thaw damage by rendering the C-S-H surface hydrophobic, for instance by using carbon particles [START_REF] Pellenq | Electron conducting carbon-based cement[END_REF]. A similar approach could be designed for the mitigation of ASR damages since the presence of ions near the gel is herein identified as a factor of risks. Depleting the interlayer of water and alkali could be done similarly by rendering the surface hydrophobic.

III. CONCLUSION

The atomistic structure of ASR gels has been obtained for the first time from a solution in which silicates are all in a monomeric form. For this, the brief-PT method, which is an accelerated molecular dynamics method, has been performed. The structure that has been formed for wet ASR gel indicates that it is a soft material with low mechanical strength, which contradicts the common scenario in which the ASR gel is growing and pressures the cement pore walls leading to irreversible damages.

We have analyzed other mechanisms that can harden the ASR gel or clog the cement pore network and we have shown that both drying and calcification effects lead to a strengthening of the ASR gel that can be one of the causes of the formation of cracks in cement. These results are in agreement with mechanical tests and structure analysis experiments.

Finally, we have revealed that when the calcification occurs, alkali ions are released in the pore solution but they remain near the ASR gel. Therefore, the interactions between the ASR gel and the C-S-H gel are modified. This is another cause that can lead to the appearance of irreversible damages in cement concrete by pressuring the cement pore wall by the intermediate of an ionic-rich solution. This indicates that the ASR damage could be mitigated by removing the ions that can accumulate in between the ASR gel and the cement pore walls.

IV. ACKNOWLEDGEMENTS

FIG. 2 .

 2 FIG. 2. (a) Evolution of Q n during the simulated formation of the gel and drying. The shadow corresponds to the standard error calculated over the 500 previous steps. (b) variations ∆Q n during the formation of the gel and drying. The inset is a snapshot of the ASR gel at 40% water content after formation. It shows the highly connected silicate structure of the silicate chains.

FIG. 3 .

 3 FIG. 3. Evolution of Q n during a simulation of the calcification. Ions are being exchanged during the first 100 ps of the simulation until all ions has been exchanged. During the last 900 ps of the simulation, ions are not exchanged.

FIG. 4 .

 4 FIG. 4. During the initial steps (a) the solution contains only calcium and the gel has a composition of Na/Si = 1, Ca/Si=K/Si=0.1. The density of silicates (b) is well distributed in the box and several species has been formed. During calcification (c), the alkali ions in the solution has been replaced by Ca 2+ . The final density profile of silicates (d) shows that silicates are attracted by the ions in the solution. In the final state, (e) the gel is filled with Ca 2+ and (f) the solution is filled with Na + .

Calculations have been performed on the GENCI/CINES infrastructure with the allocation number A0090911009.


Anne Pajot
email: anne.pajot@gmail.com

Johann Lavaud²

Gregory Carrier

Thomas Lacour

Luc Marchal

Elodie Nicolau

Light-response in two clonal strains of the haptophyte Tisochrysis lutea: evidence for different photoprotection strategies

Keywords: Tisochrysis lutea, photoprotection, diatoxanthin, zeaxanthin, echinenone, lhcx

To assess the mechanisms of photoprotection in T. lutea, two clonal strains with different basal pigments composition were studied. One synthesized echinenone, while the other did not but showed a high amount of diadinoxanthin and diatoxanthin.

We investigated the photosynthetic response of these two clonal strains in turbidostat, at different growth culture irradiances from 50 to 550 µmol photons m 2 s -1 . To this end, variable chlorophyll a fluorescence, pigment composition and transcription level of specific genes were monitored. In addition to the genes coding for the Fucoxanthin Chlorophyll a, c binding Protein (FCP), we followed the expression of several putative genes coding for the diadinoxanthin deepoxidase, the violaxanthin de-epoxidase and the zeaxanthin epoxidase enzymes. It was the first time that these genes were characterized in T. lutea.

Both clonal strains decreased their photosynthetic pigments with increasing irradiance.

Nevertheless, the two clonal strains had different photoprotection strategies illustrated with the extent of the dissipation of excess light energy. It was accompanied by the synthesis of photoprotective pigments to different extents: T-5cl6 increased its pool of diadinoxanthindiatoxanthin with increasing irradiance, while T 4cl3 preferentially synthesized echinenone above a certain level of irradiance. These diverging phenotypes were correlated with variations in the expression of lhcx, lhcr, and of the putative genes of the enzymes involved in the xanthophyll pigment cycles.

Introduction

Chromista is a well-known clade responsible for 25% of the primary production on Earth [START_REF] Cavalier-Smith | Chromalveolate diversity and cell megaevolution: interplay of membranes, genomes and cytoskeleton, in: Organelles, Genomes and Eukaryote Phylogeny: An Evolutionary Synthesis in the Age of Genomics[END_REF], composed of brown seaweeds and microalgae, including diatoms and haptophytes. These latter groups are ubiquitous in oceans, and are able to cope with fluctuating light conditions. While performing photosynthesis for their growth, cells have to avoid the photodamages caused by an excess of light energy absorption, generating various reactive oxygen species (ROS) [START_REF] Telfer | Singlet Oxygen Production by PSII Under Light Stress: Mechanism, Detection and the Protective role of β-Carotene[END_REF][START_REF] Edreva | Generation and scavenging of reactive oxygen species in chloroplasts: a submolecular approach[END_REF]. The achievement of an efficient balance between photosynthesis and photoprotection explains part of the worldwide expansion of diatoms and haptophytes [START_REF] Lohman | The ubiquitous diatom -a brief survey of the present state of knowledge[END_REF][START_REF] Guiry | How Many Species of Algae Are There?[END_REF][START_REF] Edvardsen | Diversity and distribution of haptophytes revealed by environmental sequencing and metabarcoding -a review[END_REF], and they developed several strategies to achieve it. The major player of this very dynamic equilibrium is the lightharvesting antenna, also called the light-harvesting complex (LHC) [START_REF] Büchel | Light harvesting complexes in chlorophyll c-containing algae[END_REF].

The LHC of diatoms and haptophytes, common to all Chromista, is composed of photosynthetic and photoprotective pigments, and of the Fucoxanthin Chlorophyll a,c binding Protein (FCP) [START_REF] Büchel | Light harvesting complexes in chlorophyll c-containing algae[END_REF]. The FCP is a thylakoid transmembrane protein complex composed of three main protein families. The Lhcf and Lhcr are protein sequences binding mostly photosynthetic pigments such as fucoxanthin (Fx) and chlorophylls (Chl a and c). The Lhcx bind mostly photoprotective pigments such as diadinoxanthin (Ddx), diatoxanthin (Dtx) [START_REF] Beer | Subunit Composition and Pigmentation of Fucoxanthin-Chlorophyll Proteins in Diatoms: Evidence for a Subunit Involved in Diadinoxanthin and Diatoxanthin Binding[END_REF][START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF][START_REF] Lepetit | High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool[END_REF]. It is therefore assumed that Lhcf and Lhcr are related to photosynthesis and Lhcx to photoprotection. However, Lhcf can also bind Ddx, for instance in the diatom Phaeodactylum tricornutum [START_REF] Wang | Structural basis for blue-green light harvesting and energy dissipation in diatoms[END_REF], highlighting the more complex role of Lhcf within the light-harvesting antenna. Currently in the literature, there is an abundance of studies on the FCP of diatoms [START_REF] Büchel | Light-Harvesting Complexes of Diatoms: Fucoxanthin-Chlorophyll Proteins[END_REF][START_REF] Fisher | Divergence of photosynthetic strategies amongst marine diatoms[END_REF], whereas only a few exist for haptophytes [START_REF] Lefebvre | Characterization and expression analysis of the Lhcf gene family in Emiliania huxleyi (haptophyta) reveals differential responses to light and CO2[END_REF][START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF]. We selected Tisochrysis lutea as a representative of haptophyte microalgae, and determined the composition of its FCP in a previous study [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF]. [START_REF] Lacour | Diversity in Xanthophyll Cycle Pigments Content and Related Nonphotochemical Quenching (NPQ) Among Microalgae: Implications for Growth Strategy and Ecology[END_REF] Lhcf, 12 Lhcr and 12 Lhcx protein sequences constitute the FCP of T. lutea, and at least five Fx molecules and nine Chl a and c molecules are bound to a majority of Lhcf monomers in this species [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF]. In diatoms and haptophytes, a correlation was already established between the expression of the FCP and the concentration of pigments in cells. Especially, a clear correlation between the Lhcx and the photoprotective xanthophyll cycle Ddx-Dtx was observed in diatoms and haptophytes. In the diatom Thalassiosira pseudonana, both Dtx content and Lhcx proteins increased under highlight [START_REF] Zhu | Photoprotection in the diatom Thalassiosira pseudonana: Role of LI818-like proteins in response to high light stress[END_REF]. In Phaeodactylum tricornutum, the same correlation was observed between the Ddx-Dtx pool, three lhcx genes [START_REF] Lepetit | High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool[END_REF], and the Lhcx2 and Lhcx3 proteins [START_REF] Blommaert | Contrasting NPQ dynamics and xanthophyll cycling in a motile and a non-motile intertidal benthic diatom[END_REF][START_REF] Lepetit | The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis[END_REF]. In T. lutea, the increase in Dtx was also correlated with the increase in lhcx genes expression under high light during a day:night cycle experiment [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF].

Under high light conditions, Ddx and violaxanthin (Vx) are de-epoxidized into Dtx and Zx respectively. These reactions are driven by the diadinoxanthin de-epoxidase (DDE) and the violaxanthin de-epoxidase (VDE). Reverse reactions of epoxidation imply epoxidases, and take place at low light [START_REF] Müller | Non-Photochemical Quenching. A Response to Excess Light Energy[END_REF]. These enzymes are activated by the development of a light-driven proton gradient across the thylakoid membrane and an acidification of the thylakoid lumen [START_REF] Schaller-Laudel | The diadinoxanthin diatoxanthin cycle induces structural rearrangements of the isolated FCP antenna complexes of the pennate diatom Phaeodactylum tricornutum[END_REF].

Contrary to the Ddx-Dtx cycle, well-known and well-described in diatoms and haptophytes, the Vx-Zx cycle is usually associated to higher plants and green algae only [START_REF] Fufezan | Identification of Key Residues for pH Dependent Activation of Violaxanthin De-Epoxidase from Arabidopsis thaliana[END_REF][START_REF] Havaux | The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism[END_REF][START_REF] Yamamoto | Studies on the light and dark interconversions of leaf xanthophylls[END_REF]. However, this cycle is also present in brown algae, in diatoms, where it prevents photoinhibition [START_REF] Kuczynska | Photosynthetic Pigments in Diatoms[END_REF][START_REF] Kuczynska | The xanthophyll cycle in diatom Phaeodactylum tricornutum in response to light stress[END_REF], and haptophytes such as T. lutea, as a secondary xanthophyll cycle [START_REF] Lohr | Algae displaying the diadinoxanthin cycle also possess the violaxanthin cycle[END_REF][START_REF] Gonçalves De Oliveira-Júnior | Updated pigment composition of Tisochrysis lutea and purification of fucoxanthin using centrifugal partition chromatography coupled to flash chromatography for the chemosensitization of melanoma cells[END_REF]. Together with Lhcx proteins, both Ddx-Dtx and Vx-Zx cycles participate in the non-photochemical quenching (NPQ), more precisely the fast energy-dependent quenching (qE), which allows the dissipation, as heat, of excess excitation energy, induced by high irradiance [START_REF] Malnoë | Photoinhibition or photoprotection of photosynthesis? Update on the (newly termed) sustained quenching component qH[END_REF]. In Chromista, models for NPQ measurement were established mainly for diatoms, and are not perfectly adapted to haptophytes, including T. lutea [START_REF] Lacour | Diversity in Xanthophyll Cycle Pigments Content and Related Nonphotochemical Quenching (NPQ) Among Microalgae: Implications for Growth Strategy and Ecology[END_REF]. Indeed, the NPQ of T. lutea is sustained, which means NPQ is still present in darkness, i.e. its relaxation kinetics is not minutes but hours [START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF][START_REF] Lavaud | The Peculiar Features of Non-Photochemical Fluorescence Quenching in Diatoms and Brown Algae, in: Non-Photochemical Quenching and Energy Dissipation in Plants[END_REF].

Therefore, the usual NPQ measure is not reliable for T. lutea. To overcome this problem, and in order to evaluate the dynamics between photochemistry and photoprotection, the measurement of the dark relaxation of the maximum quantum yield of the PSII (Fv/Fm) on steady-state photoacclimated microalgal cultures is required [START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF]. In these specific conditions, and as shown in diatoms, Fv/Fm is related to the NPQ extent and to the Dtx amount [START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF] In this work, two clonal strains of T. lutea were selected for their different pigment composition. Indeed, it was previously observed that T-5cl6 accumulated more Dtx and Vx than T-4cl3, and that T-4cl3 produced 19 times more echinenone than T-5cl6 under N-deprivation condition (Pajot et al., in prep.). The overall objective of this study was to understand how potentially different photoprotection strategies would be expressed in these two strains. Using turbidostat cultures, we examined in both strains the photoacclimative response (pigments and transcriptomics) and the extent of the dissipation of the excess light (dark Fv/Fm as a proxy for NPQ) as a function of increasing irradiance. The genes of interest we investigated were the genes coding for the light-harvesting antenna proteins (lhcf, lhcx and lhcr), and for the deepoxidases and epoxidases involved in the xanthophyll pigment cycles (DDE, VDE, ZE).

Materials and Methods

Culture conditions

Experiments were performed with two Tisochrysis lutea clonal strains, T-5cl6 and T-4cl3, respectively clone 6 from strain Ifremer-Argenton, isolated in Atlantic Ocean near Argenton, France and clone 3 from strain RCC1344, isolated in Atlantic Ocean at the level of the spanish coast. These strains were polyclonal and the different clones have been isolated by flow cytometry (platform Cytocell, University of Nantes, France). Current analyses performed in the laboratory are demonstrating genetic polymorphism between the two strains, resulted in phenotypic differences at the pigments and lipids content level.

We set up an experiment to observe the physiological response of the two strains with varying growth irradiances. Inoculum were maintained in Walne's medium (Walne, 1966) at 150 μmol photons m -2 s -1 (or PPF: Photosynthetic Photon Flux). The effect of different light irradiances [START_REF] Pfundel | The pH Dependence of Violaxanthin Deepoxidation in Isolated Pea Chloroplasts[END_REF]150,300 and 550 μmol photons m -2 s -1 ) was determined for each strain in continuous 3.5 L photobioreactors (PBR). These PBR were made of two transparent polymethylmetacrylate (PMMA) columns (60 mm diameter) connected by two flanges (for design reference, see the single module in Loubière et al., 2009 [31]). Light was delivered by six dimmable fluorescent white tubes. It was measured outside the PBR, at middle height between the two columns, using a spherical quantum sensor (LI-250 light meter, LI-COR, 3mm diameter).

Cultures were constantly aerated and thermoregulated at 26 ± 1°C by air conditioning and water circuit. pH was maintained constant at 8.2 with automatic injections of CO2, measured with a pH measurement loop (electrode Inpro 4800/225/PT1000, Mettler Toledo and HPT 63, LTH electronics Ltd). Walne's medium enriched sea water (1 mL L -1 ) was provided automatically by a metering pump (Simdos®) in order to maintain the culture at constant turbidity, i.e. at constant cell concentration. The excess culture was eliminated through an overflow pipe into a container. Cultures were monitored by a sensor to be maintained at low turbidity, between 1.5 and 2.5.10 6 cells mL -1 (Fig. S4) in order to reduce self-shading and therefore keep the optical properties of the cultures stable. Each experiment was conducted in triplicate. Small error bars confirm the relevance of the three replicates in validating the biological data (Fig. S4). Cultures were maintained seven to ten days at each light irradiance before sampling, in order for cells to acclimate and cultures to be at the equilibrium, i.e. at a steady-state. Cultures were assumed to be at steady-state when Chl a content was stable for at least three consecutive days with less than 10 % variation (Fig. S5,S6). In total, the experiment lasted four to five weeks per clonal strain.

Chl a, cell concentration monitoring and growth rate

To measure the Chl a content, cells were harvested daily from 10 mL of culture on a 0.2 µm fiberglass filter, and immediately immersed in 1.5 mL 95% acetone, during 24 hours.

Absorbance was measured at 665 and 750 nm, before and after acidification with HCl 0.3 M.

The number of cells per mL of culture (cell concentration, Fig. S4) was measured on a Multisizer Counter Coulter (Beckman Coulter®). Growth rate (µ) was measured daily by weighing the excess culture in the overflow container, which represented the daily dilution rate of the culture (Fig. S4).

Pigment extraction and HPLC analysis

Cell pigment content (Chl a in pg cell -1 , Fx, Ddx, Dtx and echinenone in mol(100 mol Chl a) -1 was measured after seven to ten days at each light irradiance (50, 150, 300 and 550 PPF). Cells were harvested on a 0.2 μm fiberglass filter (GF/F, Whatman TM ) and immediately stored at -80°C. Four weeks later, they were immersed in 2 mL of 95% acetone, subjected to an ultrasonic bath for 10 minutes, and placed at -20°C overnight. The acetone extracts were filtered on a 0.2 μm fiberglass filter prior to injection in HPLC. The filtered acetone extracts were analyzed by HPLC-UV-DAD (Agilent Technologies series 1200 HPLC-UV-DAD) using an Eclipse XDB-C8 reverse phase column (150 x 4.6 mm, 3.5 μm particle size, Agilent) following the method described by Van Heukelem & Thomas (2001) [START_REF] Van Heukelem | Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments[END_REF] with slight modifications. Briefly, solvent A was 70:30 MeOH: H2O 28 mM ammonium acetate and solvent B was pure MeOH (Merck France). Gradient elution was the same as described in Van Heukelem & Thomas (2001) [START_REF] Van Heukelem | Computer-assisted high-performance liquid chromatography method development with applications to the isolation and analysis of phytoplankton pigments[END_REF].

Quantification was carried out using external calibration against pigment standards (DHI, Denmark).

Chlorophyll fluorescence measurement

At steady-state, the triplicate culture subsamples acclimated to each growth irradiance were harvested. In parallel with pigment content, the chlorophyll fluorescence was measured with a PHYTO-PAM (WALZ). The effective PSII quantum yield in the dark ( / ) was measured immediately after sampling, and following 15 min, 1 h and 2 h of dark acclimation, and calculated as:

/ = _
With F0 and Fm the minimal and maximal fluorescence respectively.

RNAseq

At the steady-state, 50 mL of triplicate cultures acclimated to each growth irradiance were sampled for transcriptomic analysis. After centrifugation (10 min, 8000 g), total RNA was extracted from each strain (T-5cl6 and T-4cl3) using the TRIZOL reagent (Invitrogen, USA)

according to the manufacturer's instructions. DNase treatment (DNase RQ1, Promega) was used to remove residual genomic DNA. The quantity of purified total RNA was determined by a Qubit 3 Fluorometer (Invitrogen, USA) using the AccuBlue® Broad Range RNA Quantitation Kit (Biotium, USA). The quality of purified total RNA was determined by measurement of absorbance (260 nm/280 nm) using a Nanodrop ND-1000 spectrophotometer (LabTech, USA).

Poly(A) mRNA were selected, libraries of RNAseq were built using bare code according to Illumina's protocol. Sequencing was performed using the paired-ends method with an Illumina NovaSeq sequencer by the GenoToul platform (INRAE, Toulouse, France). The read length was 260 bases. Transcripts were normalized according to the median of ratio method proposed by the DESseq2 package in R: counts were divided by sample-specific size factors determined by the median ratio. The median ratio is the ratio of gene counts relative to geometric mean per gene. Table 1 shows the average number of normalized transcripts for each strain at each growth irradiance. Raw reads of each sample were filtered using TrimGalore to remove known Illumina adapter sequences. Low quality reads were excluded using a quality score threshold of 30 and a minimal length of 75 or 150 bases. The quality of reads was assessed using FastQC. Then, sequenced reads for each sample were aligned using HISAT2. Each gene within the alignment was counted using the Rsubread package in R with the function featureCounts, based on T. lutea reference genome [START_REF] Berthelier | Annotation of the genome assembly (version 2) of the microalga Tisochrysis lutea[END_REF]. Gene counts were obtained for each sample and normalized with DESeq2.

Similarity research

We performed a similarity research using BLAST [START_REF] Altschul | Basic local alignment search tool[END_REF] between the annotated DDE, VDE, VDL (violaxanthin de-epoxidase like) and VDR (violaxanthin de-epoxidase related) of several diatoms and haptophytes and T. lutea genome (Table S1 In haptophytes, there were seven ZE from C. tobinii and E. huxleyi.

Statistics

For each average measure of the culture triplicates (pigment content and normalized transcript counts), we used a confidence interval, characterized by error bars on graphics, calculated at a 95% confidence level:

Confidence interval = / * ( )
With / = 1,96, = standard deviation, = 3 (number of triplicates)

Statistical differences were calculated between T-5cl6 and T-4cl3. The p-values indicated in

each figure is therefore representative of the difference between the two strains (except in Fig. 6). At each irradiance, the dataset was made of three values, corresponding to the three culture replicates of T-5cl6 or T-4cl3. Therefore, a Student test was carried out first, and then an ANOVA test.

Results

Photosynthetic pigments and photochemical properties

Chl a content per cells gradually decreased with growth irradiance for both strains (Fig. 1A).

In both strains, Fx normalized by Chl a gradually decreased with growth irradiance (Fig. 1B).

At 50 PPF, T-5cl6 contained more Chl a than T-4cl3, and more Fx, as the Fx per Chl a ratio was similar in both strains. At 550 PPF it was the contrary, T-5cl6 contained less Chl a than T-4cl3.

In both strains, Fv/Fm decreased with the increase of growth irradiance (Fig. 1C). For all growth irradiances, Fv/Fm in T-5cl6 was lower than in T-4cl3. The difference between the two strains was the most significant at 150 PPF (pvalue < 0.01).

Photoprotective pigments and Fv/Fm

With increasing growth irradiance, the Ddx+Dtx pool size increased 4-fold in T-5cl6 and 2.75fold in T-4cl3 (Fig. 2A). The difference between the two strains was mainly explained by the highly variable values of Dtx amount (Fig. 2B) which increased 30.5-fold in T-5cl6 and 25.5fold in T-4cl3 between 50 and 550 PPF. Furthermore, at 50, 150, 300 and 550 PPF, there was respectively 2, 4, 3 and 2.5 more Dtx in T-5cl6 than in T-4cl3. In parallel, the de-epoxidation state of Ddx to Dtx (DES-DD), expressing the conversion of Ddx into Dtx as a photoprotection mechanism, was calculated (Fig. 2C). At the lowest irradiance of 50 PPF, the DES-DD was 4%

for T-4cl3 and significantly higher for T-5cl6, 9%. For both strains the DES-DD increased with increasing growth irradiance, and values were higher for T-5cl6 at all four growth irradiances.

At the highest irradiance, 550 PPF, the DES-DD reached 66% for T-5cl6 and 40% for T-4cl3 (Fig. 2C).

The maximum PSII quantum yield (Fv/Fm) was expressed as a function of the normalized amount of Dtx to Chl a (Fig. 2D). Trend lines of both strains have a R² > 0.97, which demonstrated a solid correlation between Fv/Fm and Dtx (Fig. 5D). The slope of the T-4cl3 trend line was higher than the one of T-5cl6. The Vx+Zx pool size increased with increasing growth irradiance, and values for T-5cl6 were significantly higher than for T-4cl3 (Fig. 3A). As for Dtx, Zx was significantly higher in T-5cl6 from 150 to 550 PPF than in T-4cl3 where Zx was synthetized in small amounts at 550 PPF only (Fig. 3B). Values of the de-epoxidation state from Vx to Zx (DES-VZ) showed that in T-5cl6, Vx was converted into Zx gradually with increasing growth irradiance, and reached 50% at 550 PPF (Fig. 3C). In T-4cl3, only 9% of Vx was converted into Zx at 550 PPF. Fv/Fm was expressed relatively to the photoprotective pigment Zx (Fig. 3D). The slope of T-4cl3 was higher than the one of T-5cl6, it was associated with 6.5-fold less Zx than in T-5cl6. In our growth conditions, the pigment echinenone was found only in T-4cl3 strain at 300 PPF and 550 PPF, and was increasing with growth irradiance (Fig. 4). At 550 PPF, echinenone represents 17% of the total carotenoids in T-4cl3 and 24% of the Chl a amount.

Expression of lhc genes

We observed the evolution of the lhcx expression in both strains under the four growth irradiances (Fig. 6, Fig. S1). The heatmap in Fig. 6 shows the expression level of lhcx genes and the significant difference between the four growth irradiances for both strains. Transcripts amounts of lhcx2 were by far the highest especially at 50 PPF in T-4cl3, followed by lhcx1 transcripts accounts. In T-4cl3, 8 lhcx upon 12 were remarkably and significantly upregulated at 150 PPF (lhcx1, lhcx3, lhcx4, lhcx8, lhcx9, lhcx10, lhcx11, lhcx12). Overlooking the upregulation at 150 PPF, lhcx1, lhcx3, lhcx4, lhcx8 and lhcx11 were gradually upregulated with increasing growth irradiance in both strains, up to five to ten-fold. At the highest growth irradiance (550 PPF), these genes were more expressed in T-5cl6 than in T-4cl3.

In T-4cl3, lhcx2 and lhcx7 were upregulated at 50 PPF and lhcx5 was downregulated at 50 PPF.

On the contrary, lhcx2, lhcx5 and lhcx7 were gradually downregulated with increasing growth irradiance in T-5cl6.

In T-4cl3 among the 12 lhcr, 10 were downregulated from 150 PPF to 550 PPF (Fig. S2), their expression was minimum at 50 PPF (Fig. S2). In T-5cl6, a similar pattern was observed, 10 lhcr were downregulated with increasing growth irradiance from 50 to 550 PPF (all but lhcr5 and lhcr6) (Fig. S2). In T-4cl3, lhcr5 and lhcr6 were upregulated only under 150 PPF (Fig. S2), like the majority of lhcx (Fig. 6), while in T-5cl6 they were upregulated with increasing growth irradiance. None of the lhcf genes were remarkably upregulated at 150 PPF (Fig. S3). In T-4cl3, the decreasing pattern with increasing growth irradiance from 50 to 550 PPF was only obvious for lhcf13, lhcf21, lhcf24, lhcf26 and lhcf27. In T-5cl6, all 28 lhcf were downregulated with increasing growth irradiance (Fig. S3).

Expression of de-epoxidase and epoxidase genes

Diadinoxanthin de-epoxidase, DDE

The similarity research with the DDE of T. pseudonana as a query resulted in four T. lutea genes, with the best similarity score for TISO_37047 (259) (Table S1). Furthermore, TISO_37047 had lower similarity scores with all other VDE, VDL (violaxanthin de-epoxidase like) or VDR (violaxanthin de-epoxidase related) of diatoms than with the DDE of T. pseudonana, although it had higher scores with a VDE domain-containing protein of the haptophyte E. huxleyi. Considering the fact that no DDE was annotated yet for haptophytes, and that VDE and DDE domains might resemble, TISO_37047 was the best candidate as a putative DDE enzyme. We decided to annotate this gene as DDE_L (DDE_like) in T. lutea.

According to the Interproscan analysis of this sequence, DDE_L possesses several VDE domains (Table S5). We noticed that in all Interproscan database, there was no DDE domain referenced. We monitored the evolution of DDE_L expression as a function of growth irradiance (Fig. 7). DDE_L expression in T-5cl6 was higher than in T-4cl3 under all four growth irradiances, and significantly at 50, 300 and 550 PPF. At 50 and 550 PPF, DDE_L was 3.5 times more expressed in T-5cl6 (p-value < 0.01), and transcripts accounts at these two irradiances were similar in both strains respectively. More generally, in T-5cl6, DDE_L expression increased gradually from 150 to 550 PPF while in T-4cl3 transcripts accounts were similar at 50, 300 and 550 PPF and maximum at 150 PPF (Fig. 7). No Dtx-epoxidase were found in the UniprotKB database. The similarity research with the VDE, VDL and VDR of diatoms and haptophytes as a query resulted in five T. lutea genes. The same five genes were found throughout the similarity research with diatoms and the one with haptophytes. According to Interproscan analysis of the molecular function, all five genes possess a VDE activity (Table S5). The best similarity scores were obtained with haptophyte sequences. TISO_01606 especially obtained the highest similarity score of all sequences, with the VDE2 sequence of E. huxleyi (600, Table S2).

TISO_01606 was named VDE_L1 (VDE_Like1) and considered as coding for a putative VDE enzyme. The alignment of VDE_L1 of T. lutea and VDE2 of E. huxleyi resulted in 67.4% similarity. Interestingly, VDE_L1 expression was significantly higher in T-5cl6 than in T-4cl3 at 50, 150 and 300 PPF but not at 550 PPF (Fig. 8). In T-5cl6, VDE_L1 increased from 50 to 150 PPF then stabilized at 300 PPF and decreased at 550 PPF. TISO_02731 showed high similarity score with the VDE of C. tobinii and a VDE domain-containing protein of E. huxleyi (respectively 418 and 400) (Table S2), and as such was considered as coding for a putative VDE enzyme. TISO_02731 was named VDE_L2 (for VDE_Like2). VDE_L2 expression was higher in T-5cl6 than in T-4cl3 under all growth irradiances, and significantly at 50, 150 and 300 PPF (Fig. 8). In both strains, but especially in T-5cl6, its expression decreased with increasing irradiance. According to Interproscan analysis (Table S5), VDE_L2 was associated with two biological processes, potassium ion transport and transmembrane transport, and two molecular functions, voltage-gated potassium channel activity and VDE activity. VDE_L2 was the only gene which was not exclusively associated to VDE activity among the five genes found with the similarity research. The similarity research with the ZE of diatoms and haptophytes as a query resulted in eleven T.

lutea genes (Table S3, S4). Among them, TISO_30349 had the highest similarity score with a ZE in E. huxleyi (578) (Table S4). TISO_30349 was therefore considered as coding for a putative ZE enzyme and named ZE_L (for ZE_Like). With increasing growth irradiance, the expression of ZE_L decreased in both strains (Fig. 9). At 150 and 300 PPF, ZE_L was significantly more expressed in T-4cl3.

Discussion

Different photoprotection strategies in T. lutea T-5cl6 and T-4cl3 strains

The Ddx-Dtx and Vx-Zx xanthophyll cycles

As cells maintained growth efficiency under light stress, as shown with the increasing growth rate in Fig. S4, the observed molecular events such as photoprotective pigments synthesis really

The remarkable presence of echinenone in T-4cl3

The most noteworthy result of this study was the appearance of echinenone in T-4cl3 cells at 300 PPF. The presence of echinenone was previously demonstrated in T. lutea strain CCAP 927/14 [START_REF] Mulders | Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO[END_REF] and in a close species, Isochrysis galbana, under N-deprivation [START_REF] Mulders | Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO[END_REF][START_REF] Flynn | Changes in carbon and nitrogen physiology during ammonium and nitrate nutrition and nitrogen starvation in Isochrysis galbana[END_REF]. In addition, we observed that T-4cl3 also synthetized echinenone under N-deprivation (Pajot et al. in prep).

In T. lutea strain CCAP 927/14, it was reported that echinenone represented 25% of total carotenoids under N-deprivation [START_REF] Mulders | Growth and pigment accumulation in nutrient-depleted Isochrysis aff. galbana T-ISO[END_REF]. Our results showed that echinenone represented a similar high amount of 17% of total carotenoids under N-replete condition at high irradiance.

Echinenone is usually found in cyanobacteria, it scavenges the ROS [START_REF] Takaichi | Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotenoid glycosides[END_REF] under high irradiance, and as such participates to photoprotection. In cyanobacteria, under high irradiance, the thermal dissipation of the light energy absorbed in excess by the phycobilisome (the light-harvesting antenna of cyanobacteria) is also mediated by a NPQ mechanism, which regulatory partners are different from haptophytes and diatoms [START_REF] Punginelli | Influence of zeaxanthin and echinenone binding on the activity of the Orange Carotenoid Protein[END_REF]. Cyanobacteria NPQ occurs when the echinenone is bound to the Orange Carotenoid Protein (OCP) [START_REF] Wilson | A Soluble Carotenoid Protein Involved in Phycobilisome-Related Energy Dissipation in Cyanobacteria[END_REF][START_REF] Holt | A carotenoid-protein from cyanobacteria[END_REF]. For instance, in Synechocystis sp., NPQ is only activated when the 3′-hydroxyechinenone is bound to the OCP [START_REF] Kusama | Zeaxanthin and Echinenone Protect the Repair of Photosystem II from Inhibition by Singlet Oxygen in Synechocystis sp. PCC 6803[END_REF]. Our work is a first report of the presence of echinenone in a haptophyte under high light. According to several similarity researches we performed, no OCP was found in T. lutea. However, our results support that in the strain T-4cl3, the echinenone played a role in the response to high light as a support to the xanthophyll cycles-mediated NPQ, likely through direct ROS scavenging [START_REF] Mohamad | Singlet oxygen quenching by thione analogues of canthaxanthin, echinenone and rhodoxanthin[END_REF],

i.e. by free-echinenone molecules in the thylakoid membrane, similarly as some Dtx molecules [START_REF] Lepetit | Evidence for the Existence of One Antenna-Associated, Lipid-Dissolved and Two Protein-Bound Pools of Diadinoxanthin Cycle Pigments in Diatoms[END_REF]. Nevertheless, we cannot exclude that a different form of the OCP is present in T. lutea, and further search is required.

Xanthophyll de-epoxidases and epoxidases in T. lutea

This is the first time that several DDE and VDE candidate genes were characterized in T. lutea.

The monitoring of their expression as a function of growth irradiance enabled to correlate the transcription of these genes to the different light-response strategies shown by T-5cl6 and T-4cl3. No Dtx-epoxidase was found in the UniprotKB database. However, the two Dtxepoxidases of Thalassiosira pseudonana are usually annotated as ZE in the literature [START_REF] Goss | Biodiversity of NPQ[END_REF].

Therefore, the seven sequences in T. lutea resulting from the research similarity with the query sequence "ZE Thalassiosira pseudonana THAPSDRAFT 261390" (Table S3) might be good candidates as Dtx-epoxidase.

As pointed out before in the diatom P. tricornutum, the zeaxanthin-epoxidase (ZE) can play an equivalent role, i.e. ZE can trigger the epoxidation of both Dtx and Zx [START_REF] Lavaud | The Peculiar Features of Non-Photochemical Fluorescence Quenching in Diatoms and Brown Algae, in: Non-Photochemical Quenching and Energy Dissipation in Plants[END_REF][START_REF] Kusama | Zeaxanthin and Echinenone Protect the Repair of Photosystem II from Inhibition by Singlet Oxygen in Synechocystis sp. PCC 6803[END_REF].

The diadinoxanthin de-epoxidase

With increasing growth irradiance, Ddx was de-epoxidized into Dtx as illustrated by the progressive increase in DES-DD in both strains. Ddx de-epoxidation was stronger in T-5cl6 than in T-4cl3. We found a DDE gene candidate we named DDE_L, whose associated protein could be responsible of the Ddx de-epoxidation in T. lutea. As for Dtx content and DES-DD, DDE_L expression was higher in T-5cl6 than in T-4cl3 under all four irradiances, which was consistent with a stronger Ddx de-epoxidation activity in T-5cl6. Interestingly, in T-5cl6, the expression of DDE_L was close to its maximum already at 50 PPF. In parallel, we did not observe a particular Dtx accumulation that could have been induced by chlororespiratory electron flow, as it was reported in the diatom Phaeodactylum tricornutum [START_REF] Jakob | Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum[END_REF]. This result might suggest that T-5cl6 cells produce a maximum of DDE_L transcripts regardless of the irradiance, without translating them into protein unless it is necessary. Or, the associated protein of DDE_L has a more complex role than the putative de-epoxidation of Ddx. In P. tricornutum, the inactivation of the DDE enzyme under low light was shown not to be the factor triggering the inactivation of Dtx synthesis and NPQ maintenance [START_REF] Blommaert | The fine-tuning of NPQ in diatoms relies on the regulation of both xanthophyll cycle enzymes[END_REF]. Instead, they were due to an increase in Dtx epoxidation, which was therefore proposed to be the main actor in the Ddx-Dtx xanthophyll cycle regulation.

The violaxanthin de-epoxidase and the zeaxanthin epoxidase

The difference between the two clonal strains was also witnessed by the expression level of the Vx-Zx cycle associated genes. Among them, the expression of TISO_01606, we named VDE_L1, was significantly higher in T-5cl6 at almost all growth irradiances, and it increased with increasing growth irradiance. A second putative candidate, TISO_02731, we named VDE_L2, was found to be associated to both a VDE activity and a voltage-gated potassium channel protein. A voltage-gated potassium channel protein is a complex forming a transmembrane channel through which potassium ions may cross a cell membrane in response to changes in membrane potential [START_REF] Yellen | The voltage-gated potassium channels and their relatives[END_REF]. It is noteworthy that VDE_L2 could regulate in parallel both the de-epoxidation of Vx and the pH lumen which is itself pH-dependent [START_REF] Fufezan | Identification of Key Residues for pH Dependent Activation of Violaxanthin De-Epoxidase from Arabidopsis thaliana[END_REF][START_REF] Pfundel | The pH Dependence of Violaxanthin Deepoxidation in Isolated Pea Chloroplasts[END_REF]. Such potential dual role would need further investigation, ideally with targeted knock-out mutagenesis, as in diatoms [START_REF] Lavaud | Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching[END_REF]. Furthermore, VDE_L1 in T. lutea had the best similarity score with the VDL2 gene of P. tricornutum (gene identification: PHATRDRAFT_45846), which encodes for an enzyme that has recently been demonstrated to be central in the Fx biosynthetic pathway in a study by [START_REF] Bai | Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin[END_REF] [START_REF] Bai | Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin[END_REF]. This study suggested that Ddx would also be a substrate for the VDL2 enzyme, in addition to Vx. VDL2 would indeed be able to catalyze the tautomerization reaction (isomerization of a molecular function) of Ddx to allenoxanthin, these two pigments being key precursors of the Fx synthesis [START_REF] Bai | Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin[END_REF]. In addition, it is specified that the VDL2 enzyme in P. tricornutum is most likely subject to strict regulation in diatoms and haptophytes, so that Ddx can play both its role as a precursor of the major photosynthetic pigment that is Fx, and its role in photoprotection within the Ddx-Dtx cycle.

In the diatom P. tricornutum, NPQ results from the early inhibition of ZE rather than from an activation of DDE/VDE, which increases the amount of Dtx and, under certain light conditions, of Zx [START_REF] Blommaert | The fine-tuning of NPQ in diatoms relies on the regulation of both xanthophyll cycle enzymes[END_REF].The epoxidation is the reverse reaction of the Ddx-Dtx and Vx-Zx cycles and it usually corresponds to a decrease in light intensity. In T. lutea, ZE_L gene seems to be a strong candidate to code for a zeaxanthin epoxidase. In both strains, the ZE-L transcript level was similar to the ones of DDE and VDE, it was the highest at 50PPF, and its expression decreased with increasing growth irradiance, and might correspond to a lower synthesis of the ZE_L associated protein. This pattern is usual and might be interpreted as the need of cells for keeping the right balance between light-harvesting (Ddx and Vx) and photoprotective (Dtx and Zx) pigments as a function of the growth irradiance. In the study by [START_REF] Bai | Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin[END_REF] [START_REF] Bai | Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin[END_REF], the ZEP1 (zeaxanthin epoxidase) gene in P. tricornutum (gene identification: PHATRDRAFT_45845) has been shown, as VDL2, to code for a central enzyme in the Fx biosynthetic pathway [START_REF] Bai | Green diatom mutants reveal an intricate biosynthetic pathway of fucoxanthin[END_REF].

Since ZE_L in T. lutea had a high similarity score with ZEP1, it is assumed that its role, in addition to the epoxidation of Zx, is also central in the biosynthesis of Fx in T. lutea and in haptophytes in general.

A sustained NPQ in T. lutea

Culture samples were exposed to darkness in order to observe the relaxation dynamics from photoprotection back to photochemistry. Surprisingly, after two hours of darkness exposure, there was still Dtx present in cells of both strains, indicating that the epoxidation reaction from Dtx to Ddx was not complete. It has been determined that in algae using the Ddx-Dtx cycle as a photoprotective mechanism, the switch of the light-harvesting antenna from the NPQ mode to the light-harvesting mode at lower irradiance can only be realized by an efficient removal of Dtx [START_REF] Goss | The importance of a highly active and ΔpH-regulated diatoxanthin epoxidase for the regulation of the PS II antenna function in diadinoxanthin cycle containing algae[END_REF]. Nevertheless, under some harsh conditions (very high light, low temperature), the presence of Dtx, even after prolonged darkness, i.e. several hours and days, can be 'constitutive'

and can generate a sustained form of NPQ [START_REF] Lacour | Diversity in Xanthophyll Cycle Pigments Content and Related Nonphotochemical Quenching (NPQ) Among Microalgae: Implications for Growth Strategy and Ecology[END_REF][START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF]. This form of NPQ is complementary to the main NPQ component, qE [START_REF] Lacour | Diversity in Xanthophyll Cycle Pigments Content and Related Nonphotochemical Quenching (NPQ) Among Microalgae: Implications for Growth Strategy and Ecology[END_REF][START_REF] Goss | Biodiversity of NPQ[END_REF][START_REF] Niyogi | Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis[END_REF], which is usually turned off in a few tens of seconds to some minutes when the cells are transferred from excess light to lower light or darkness [START_REF] Lepetit | High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool[END_REF].

In T. lutea, the retention of Dtx under prolonged darkness was especially strong in T-5cl6 under the highest irradiances. It was correlated with a partial relaxation on Fv/Fm likely illustrating a strong sustained NPQ [START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF]. It is noteworthy that T-5cl6 is the strain which relies the most on xanthophyll cycles. A stronger sustained NPQ is likely to be part of its photoprotective strategy, allowing the cells to promptly respond to an anticipated increase in irradiance after the darkness period [START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF].

4.4.The role of lhcx in T. lutea

lhcx1, lhcx4, lhcx8 and lhcx11 as major actors in regulating NPQ?

The increase of photoprotective pigments such as Dtx and Zx does not necessarily lead to higher effective NPQ. Indeed in diatoms, it is the binding of photoprotective pigments to Lhc proteins, especially Lhcx, that effectively involves these pigments in NPQ [START_REF] Lepetit | High Light Acclimation in the Secondary Plastids Containing Diatom Phaeodactylum tricornutum is Triggered by the Redox State of the Plastoquinone Pool[END_REF]. Dtx molecules can be freely present in the thylakoid membrane to directly scavenge ROS species [START_REF] Lepetit | Evidence for the Existence of One Antenna-Associated, Lipid-Dissolved and Two Protein-Bound Pools of Diadinoxanthin Cycle Pigments in Diatoms[END_REF]. In both T. lutea strains, lhcx1, lhcx4, lhcx8 and lhcx11 were gradually upregulated with increasing growth irradiance together with Dtx synthesis. It suggests that lhcx1, lhcx4, lhcx8 and lhcx11 could be Dtx binders and potentially major actors in regulating NPQ in T. lutea. Interestingly, the transcript level of these genes was not particularly different between both strains (but for 150 PPF, see below) although NPQ was significantly stronger in T-5cl6, with the only exception of lhcx11 at 550 PPF for which there was no difference in NPQ. Therefore, the NPQ difference can only be explained by the highest transcription of lhcx genes in corresponding proteins in T-5cl6 for providing the necessary binding sites to the significantly higher amount of Dtx molecules in this strain. Western-blot analysis will be needed to further confirm this hypothesis [START_REF] Lepetit | The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis[END_REF][START_REF] Buck | Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum[END_REF]. Besides lhcx, two lhcr genes, lhcr5 and lhcr6, were upregulated with increasing growth irradiance in both strains. Interestingly, lhcr5 and lhcr6 form, together with lhcr10, a subclade of the lhcr family in T. lutea [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF]. Moreover, lhcr5 and lhcr6 are similar to lhcr6 and lhcr8 in P. tricornutum, which were proposed to play a role in photoprotection [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF][START_REF] Nymark | An Integrated Analysis of Molecular Acclimation to High Light in the Marine Diatom Phaeodactylum tricornutum[END_REF]. It is therefore consistent that lhcr5 and lhcr6 were expressed similarly to the majority of lhcx in T-4cl3. This result confirmed their supposed function in photoprotection, and possibly in NPQ.

lhcx2 and lhcx5 prevent an increase in irradiance?

Among lhcx genes, lhcx2, lhcx5 and lhcx7 were the only genes downregulated with increasing growth irradiance. In particular, lhcx2 was highly expressed at 50 PPF in T-4cl3. Similarly, in our previous study on T. lutea strain CCAP 927/14, lhcx2 was the only gene upregulated at low light, and during the night [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF]. The hypothesis was that the associated Lhcx2 protein could protect from the return of light at any time after prolonged darkness exposure, as proposed for Lhcx4 in the diatom P. tricornutum [START_REF] Taddei | Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum[END_REF]. The function of lhcx2 in T. lutea strain CCAP 927/14

and T-4cl3 might therefore be extended to respond to a potential increase of irradiance during a low light period acclimation. In T-5cl6, this role might be undertaken by lhcx5 which was also highly expressed at 50 PPF compared with T-4cl3. Transcript accounts of lhcx2 and lhcx5, as well as the high light upregulated lhcx1 and lhcx4, were the highest in both strains. Thus, corresponding proteins, if translation would be paralleled, might play an important role in lightresponse and possibly be predominant in the structure of the FCP in T. lutea.

lhcx participate in the balance between photochemistry and photoprotection?

We observed a significant spike of expression for eight lhcx and two lhcr (lhcr5 and lhcr6) at 150 PPF in T-4cl3. It was not paralleled with photoprotective pigments. The increase of lhcx and lhcr expression at 150 PPF was therefore not considered as inherent to photoprotection mechanisms under to the highest irradiances. In a previous experiment, the same 8 lhcx of T. lutea strain CCAP 927/14, cultivated with a day:night cycle, were significantly upregulated at the beginning of the day, corresponding to 225 PPF, compared to midday, corresponding to 900 PPF [START_REF] Pajot | The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis[END_REF]. The hypothesis was that cells overexpressed lhcx at the beginning of the day to endorse light onset without risking photodamage. With our results in this work, the explanation could finally be different. We hypothesize that 150 PPF is a threshold between photochemistry and photoprotection for T-4cl3, i.e. that 150 PPF would be close to EK, the light saturation parameter for growth. This possibility is supported by the sharper decrease in Fv/Fm beyond 150 PPF in T-4cl3. Another possibly, not exclusive, and depending on transcript translation, is the need for specific Lhcx and/or Lhcr FCP subunits in the light-harvesting antenna, in order to perform the best adjustment between photochemistry and photoprotection at 150 PPF and beyond.

Conclusion

This study investigated the differences of photoprotection strategies between two clonal strains of the haptophyte Tisochrysis lutea. Overall, our results showed that with increasing growth irradiance, the expression of the lhcx genes increased and it was correlated with a gradual increase of the content in photoprotective pigments and of the dissipation of light energy in excess. Both strains differed with their photoprotective pigment (Table 2). T-5cl6 strain strategy was predominantly based on the Ddx-Dtx and the Vx-Zx xanthophyll cycles, while T-4cl3 strain strategy was also based on the Ddx-Dtx cycle, but to a lesser extent, and on the parallel echinenone accumulation. At higher irradiances, when both Dtx and echinenone appeared to be insufficient to endorse the excess light energy, the photoprotective pigment content was completed with the Vx-Zx cycle.

In both strains, the fine balance between photosynthesis and photoprotection was performed by the combination of (Table 2) (1) the FCP structuration with lhcx, lhcr and lhcf, (2) the modulation of the photosynthetic pigments chlorophylls and fucoxanthin, (3) the synthesis of photoprotective pigments of the Ddx-Dtx and the Vx-Zx xanthophyll cycles, alongside with the transcription of de-epoxidases and epoxidases, and of the echinenone in T-4cl3 specifically.

The differences between the two clonal strains highlighted precise correlations between these regulatory partners. We propose these differences are likely due to their adaptation to the light climate of the natural environment from where they were isolated [START_REF] Lavaud | Photoprotection capacity differs among diatoms: Possible consequences on the spatial distribution of diatoms related to fluctuations in the underwater light climate[END_REF][START_REF] Croteau | Shifts in growth light optima among diatom species support their succession during the spring bloom in the Arctic[END_REF][START_REF] Barnett | Growth form defines physiological photoprotective capacity in intertidal benthic diatoms[END_REF], and/or to the individual evolution and selection in the laboratory growing conditions. Finally, the remarkable presence of echinenone, which content was modulated by light under N-replete condition, excludes the influence of nutrients alone on its biosynthesis.

Figure 1 :

 1 Figure 1: Evolution of the Chl a content (average ± the confidence interval, pg cell -1 ) (A), of the Fx content normalized by Chl a (average ± the confidence interval, mol(100 mol Chl a) -1 ) (B), of the maximum PSII quantum yield Fv/Fm (average ± the confidence interval) (C),relatively to growth irradiance (PPF = Photosynthetic Photon Flux = µmol m 2 s -1 ). *: pvalue < 0.05, **: pvalue<0.01. *: pvalue < 0.05, **: pvalue<0.01.

Figure 2 :

 2 Figure 2: Evolution of the Ddx+Dtx pool (A) and of the Dtx (B) normalized by Chl a (average ± the confidence interval, mol(100 mol Chl a) -1 ), of the de-epoxidation state (DES) from Ddx into Dtx (C) relatively to growth irradiance (PPF = Photosynthetic Photon Flux = µmol m 2 s -1 ). (D) presents the Fv/Fm relatively to Dtx content and the significant difference between the two slopes. *: pvalue < 0.05, **: pvalue < 0.01.

Figure 3 :

 3 Figure 3: Evolution of the Vx+Zx pool (A) and of the Zx (B) normalized by Chl a (average ± the confidence interval, mol(100 mol Chl a) -1 ), of the de-epoxidation state from Vx into Zx (C) relatively to growth irradiance (PPF = Photosynthetic Photon Flux = µmol m 2 s -1 ). (D) presents the Fv/Fm relatively to Zx content and the significant difference between the two slopes. *: pvalue < 0.05, **: pvalue < 0.01.

Figure 4 :

 4 Figure 4: Evolution of the echinenone normalized by Chl a (average ± the confidence interval, mol(100 mol Chl a) -1 ) relatively to growth irradiance (PPF = Photosynthetic Photon Flux = µmol m 2 s -1 ). **: pvalue < 0.01.

Figure 5 :

 5 Figure 5: Evolution of Fv/Fm (A), of Dtx content (B) and of Ddx content (C) normalized by Chl a (average ± the confidence interval, mol(100 mol Chl a) -1 ) relatively to the time in darkness.

Figure 6 :

 6 Figure 6: Heatmap of the lhcx transcripts counts. *: pvalue < 0.05, **: pvalue < 0.01. Thick borders correspond to the highly upregulated lhcx genes at 150 PPF in T-4cl3.

Figure 7 :Figure 8 :

 78 Figure 7: Evolution of normalized transcript counts of DDE_L (average ± the confidence interval) relatively to growth irradiance (PPF = Photosynthetic Photon Flux = µmol m 2 s -1 ). *: pvalue < 0.05, **: pvalue < 0.01.

Figure 9 :

 9 Figure 9: Evolution of TISO_33049 (ZE_L) normalized transcripts counts (average ± the confidence interval) relatively to growth irradiance (PPF = Photosynthetic Photon Flux = µmol m 2 s -1 ). *: pvalue < 0.05, **: pvalue < 0.01.

  protected the cells from damage. The two T. lutea clonal strains, T-5cl6 and T-4cl3, were grown in the exact same conditions of irradiance, temperature, pH, nutrients, and at the same cell concentration. The ratio of Fx/Chl a was lower in T-4cl3 than in T-5cl6, suggesting that the light-harvesting capacity under high light was lower in T-4cl3. Using the darkness relaxation of Fv/Fm as a proxy for NPQ[START_REF] Lacour | The Role of Sustained Photoprotective Non-photochemical Quenching in Low Temperature and High Light Acclimation in the Bloom-Forming Arctic Diatom Thalassiosira gravida[END_REF], we monitored a strong correlation with the Dtx content, pointing out to an implication of Dtx in the photoprotective dissipation of excess light energy in both strains. The decrease of photochemistry in T-4cl3 was associated to 2.5-fold less amount of Dtx than in T-5cl6, suggesting differences in quenching efficiency of Dtx between the two strains. Moreover, as in diatoms, Ddx was not entirely de-epoxidized in Dtx (i.e. maximum DES was about 60%), highlighting the double role of Ddx as a photosynthetic pigment and as a precursor of Dtx[START_REF] Arsalane | Influence of the Pool Size of the Xanthophyll Cycle on the Effects of Light Stress in a Diatom: Competition Between Photoprotection and Photoinhibition[END_REF]. The same hold true for the photoprotective couple pigment Vx and Zx, as previously reported in diatoms[START_REF] Lohr | Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model[END_REF]. In T-5cl6, photoprotection was associated with a higher Dtx and Zx content than in T-4cl3, with Zx only accumulated at 550 PPF in T-4cl3. Instead, T-4cl3 produced echinenone at 300 and 550 PPF while T-5cl6 did not. In summary, T-5cl6 used the two xanthophyll cycles Ddx-Dtx and Vx-Zx, and associated NPQ, as major photoprotective mechanisms, similarly as in diatoms[START_REF] Blommaert | Contrasting NPQ dynamics and xanthophyll cycling in a motile and a non-motile intertidal benthic diatom[END_REF]. Both cycles were used to a lower extent and more sequentially by T-4cl3. In addition, T-4cl3 cells strengthened their photoprotective pigment panel by producing echinenone at a certain threshold between 150 and 300 PPF. At 550 PPF, Dtx and echinenone were probably not sufficient to endorse the excess light energy in T-4cl3 cells and they started de-epoxidizing Vx into Zx. It is likely that at higher irradiances above 550 PPF, the Zx and echinenone concentrations would increase even more.

Table 1 :

 1 Number of normalized reads for all growth irradiances for T-5cl6 and T-4cl3

		T-5cl6	T-4cl3
	50 µmol m² -1	3.3E+07	2.6E+07
	150 µmol m² s -1	3.0E+07	2.5E+07
	300 µmol m² s -1	3.2E+07	3.7E+07
	550 µmol m² s -1	2.5E+07	2.8E+07

  At 50 PPF, except for lhcf22 and lhcf23, all lhcf were more expressed in T-5cl6 than in T-4cl3. Under the other growth irradiances, almost half of lhcf (12 among 28) were more expressed in T-5cl6 than in T-4cl3.

	The expression of other genes in T-4cl3 was either
	relatively stable without significant variations, either slightly decreased from 150 PPF to 300
	and 550 PPF. Indeed, in T-4cl3, 15 lhcf among 28 were the least expressed at 50 PPF compared
	with other irradiances (lhcf1, lhcf2, lhcf3, lhcf4, lhcf5, lhcf6, lhcf7, lhcf8, lhcf17, lhcf18, lhcf19,
	lhcf20, lhcf22, lhcf25, lhcf28).

Table 2 :

 2 Summary of the pigment content, Fv/Fm, lhcx and lhcr gene expression in T-5cl6 and T-4cl3 cells under the four growth irradiances.Legend for levels: +++ (highest), ++ (high), + (moderately high), -(moderately low), --(low), 0 (absence).

			Photophysiology			Pigments			lhcx gene expression
			Dissipation of the light energy	Photosynthetic pigments	Photoprotective pigments	Actors of NPQ	Prevent an increase in irradiance
	Strain	Light	in excess	Fx	Chl a	Dtx	Zx	Echinenone	lhcx1, lhcx4, lhcx8, lhcx11, lhcr5, lhcr6	lhcx2 lhcx5
	T-5cl6		--	+++	+++	-	0	0	--	+	+++
		50 PPF
	T-4cl3		0	+++	+++	--	0	0	--	+++	-
	T-5cl6		-	++	++	+	+	0	-	+	+
		150 PPF
	T-4cl3		--	++	++	-	0	0	+++	+	++
	T-5cl6		+	++	+	++	++	0	+	-	-
		300 PPF
	T-4cl3		-	+	+	+	0	+	+	-	+
	T-5cl6		++	-	-	+++	+++	0	++	--	-
		550 PPF
	T-4cl3		++	-	-	++	+	++	++	-	+

Acknowledgements:

The authors would like to thank Gaël Bougaran, Raymond Kaas, Jean-Baptiste Bérard, for their help with the turbidostat cultures and the sensors, Elise Robert and Agathe Maupetit, for their participation in the experiments.

Data Availability Statement:

The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA787725.

Abbreviations

Funding:

The authors would like to acknowledge Ifremer and the Region Pays de la Loire (France) for the PhD grant of Anne Pajot. This work was co-funded by the SMIDAP through the TINAQUA project.

Conflicts of Interest:

The authors declare no conflict of interest.

Author contributions:

Conceptualization, A.P., E.N., T.L., J.L.; Methodology, A.P., E.N., G.C.; Formal analysis, A.P., G.C.; Investigation, A.P., J.L.; Data curation, A.P., G.C.; Writing-Original Draft preparation, A.P.; Writing-Review & Editing, J.L., E.N., G.C., T.L.; Visualization, A.P.; Supervision, E.N., L.M.; Project Administration, E.N., L.M.