Waqar Ahmad

Adnan Zahir

Muhammad Nadeem

Laurine Garros

Samantha Drouet

Sullivan Renouard

Joel Doussot

Nathalie Giglioli-Guivarc'h

Christophe Hano
email: christophe.hano@univ-orleans.fr

Bilal Haider Abbasi
email: bhabbasi@qau.edu.pk

Joël Doussot

Enhanced production of lignans and neolignans in chitosan-treated flax (Linum usitatissimum L.) cell cultures

Keywords: Linum usitatissimum, chitosan, lignans, neolignans, elicitation, cell culture

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Flax (Linum usitatissimum L.) is used for more than 10,000 years in terms of consumption and cultivation [START_REF] Helbaek | Plant collecting, dry-farming, and irrigation agriculture in prehistoric Deh Luran[END_REF][START_REF] Zeist | Paleobotanical results of the 1970 season at Cayonu, Turkey[END_REF]. More recently, the detection of polyphenols (lignans, neolignans) has opened new dimensions for flax in pharmaceutical and nutraceutical industries to be used for preventing cancers, cardiovascular disorders, diabetes, lupus nephritis and other disorders [START_REF] Westcott | Chemical studies on the constituents of Linum spp, Flax, the genus Linum[END_REF]. Along with lignans, L. usitatissimum is considered a good source for accumulation of neolignans which are potential candidates in anti-inflammatory and antifungal applications [START_REF] Anjum | Effects of photoperiod regimes and ultraviolet-C radiations on biosynthesis of industrially important lignans and neolignans in cell cultures of Linum usitatissimum L.(Flax)[END_REF]. [START_REF] Haworth | The chemistry of the lignan group of natural products[END_REF] coined the term lignan for the first time to define a class of dimeric phenylpropanoids in which two units C6-C3 are linked by the central C8 [START_REF] Haworth | The chemistry of the lignan group of natural products[END_REF], lignans are a group of phytochemicals (polyphenols) which are formed by connection of 2 cinnamyl alcohols [START_REF] Xia | Dirigent-mediated podophyllotoxin biosynthesis in Linum flavum and Podophyllum peltatum[END_REF].

Whereas [START_REF] Gottlieb | Fortschritte der Chemie Organischer Naturstoffe/Progress in the chemistry of organic natural products[END_REF] speculated that molecules having 2 phenylpropanoid parts attached in a different fashion, such as C5-C5' should be called neolignans [START_REF] Gottlieb | Fortschritte der Chemie Organischer Naturstoffe/Progress in the chemistry of organic natural products[END_REF]. Lignans and neolignans are structurally made of two propyl benzene units (Fig 1). In lignans these units are associated by a β-β′ bond while this substitution pattern is different in neolignans [START_REF] Teponno | Recent advances in research on lignans and neolignans[END_REF]. Lignans are reported in more than 65 families of vascular plants, and are extracted from almost all parts of a plant [START_REF] Gordaliza | Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives[END_REF]. Pharmacological activities of lignans have been demonstrated in various experimental models.

Lignans induce the synthesis of 2-hydroxy estrogen in females which has a potential role in deterrence of cancer [START_REF] Martin | Interactions between phytoestrogens and human sex steroid binding protein[END_REF]. Lignans are believed to be responsible for inhibiting the growth of different human prostate cancer cell lines [START_REF] Li | Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice[END_REF][START_REF] Demark-Wahnefried | Pilot study of dietary fat restriction and flaxseed supplementation in men with prostate cancer before surgery: exploring the effects on hormonal levels, prostate-specific antigen, and histopathologic features[END_REF] According to reported data [START_REF] Danbara | Enterolactone induces apoptosis and inhibits growth of Colo 201 human colon cancer cells both in vitro and in vivo[END_REF] when a dose of 10 mg/kg of lignans administered subcutaneously in athymic mice, have diminished the proliferation of 201 human colon cancer cell lines. Flax was also reported to abate the elevated blood glucose concentration in post meal duration [START_REF] Cunnane | High α-linolenic acid flaxseed (Linum usitatissimum): some nutritional properties in humans[END_REF][START_REF] Jenkins | Health aspects of partially defatted flaxseed, including effects on serum lipids, oxidative measures, and ex vivo androgen and progestin activity: a controlled crossover trial[END_REF]. A study conducted on a group of females in which they were fed with ground flax seeds 50 g/day, after four weeks their blood glucose level has been significantly reduced [START_REF] Skrzypczak-Pietraszek | The influence of L-phenylalanine, methyl jasmonate and sucrose concentration on the accumulation of phenolic acids in Exacum affine Balf. f. ex Regel shoot culture[END_REF].

The production of secondary metabolites in different plant culture techniques using biotechnological interventions is an attractive substitute to the isolation from whole plant materials [START_REF] Skrzypczak-Pietraszek | The influence of L-phenylalanine, methyl jasmonate and sucrose concentration on the accumulation of phenolic acids in Exacum affine Balf. f. ex Regel shoot culture[END_REF]. Using plant cell cultures coupled with elicitation strategies such as introduction of physical and chemical entities to the cell culture per se or growth conditions, as a biological platform for the synthesis of secondary metabolites is a promising approach for the sustainable production in modern medicinal and aromatic industry [START_REF] Murthy | Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation[END_REF].

Due to growing interests on lignans and neolignans, alternate platforms for their production other than conventional plant cultivation are needed. Cell and tissue culture techniques could be used for the enhanced biosynthesis of these metabolites. Chitosan is a polycationic β-1,4 linked Dglucosamine polymer known to act as a bioactive antifungal agent [START_REF] Hirano | Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens[END_REF] though the elicitation of pathogenesis-related proteins in the host [START_REF] Benhamou | Induction of systemic resistance to Fusarium crown and root rot in tomato plants by seed treatment with chitosan[END_REF] and stimulation of phytoalexin production [START_REF] Cuero | Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents[END_REF].

Chitosan has not been exploited previously for the accumulation of these valuable lignans and neolignans in cell cultures of flax. Therefore, in current study effect of chitosan is evaluated based on its impact on the biosynthesis and dynamics of these anti-cancer lignans and neolignans. A validated reverse-phase high performance liquid chromatography (RP-HPLC DAD) was used for the quantification of lignans and neolignans. As per our knowledge, this is the first report on enhanced biosynthesis of lignans (secoisolariciresinol diglucoside (SDG) and lariciresinol diglucoside (LDG)) and neolignans (dehydrodiconiferyl alcohol diglucoside (DCG) and guaiacylglycerol-β-coniferyl ether diglucoside (GGCG)) using chitosan as a stimulator of lignans and neolignans production in flax cell cultures. Current research has a potential to be scaled up to bioreactor levels for the feasible biosynthesis of these commercially important metabolites.

Materials and Methods

Plant materials

Flax seeds (brown variety) were collected from malakand division hills (natural habitat) Khyber Pakhtunkhwa, Pakistan. The flax cultivar Barbara was supplied by Coopérative Terre de Lin (St Pierre le Viger, France). The generation of the RNAi plants and the homozygous transgenic lines were obtained as previously described [START_REF] Renouard | RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: consequences on lignans and neolignans accumulation[END_REF].

For germination, seeds were surface sterilized using 0.1% mercuric chloride and 70% ethanol for 30 and 60 seconds respectively, subsequently washed 3 times with dH2O (autoclaved water) and dried using sterilized filter paper sheet. The sterilized seeds were inoculated on MS (Murashige and Skoog basal Media) following the protocol described earlier [START_REF] Abbasi | Differential effects of in vitro cultures of Linum usitatissimum L.(Flax) on biosynthesis, stability, antibacterial and antileishmanial activities of zinc oxide nanoparticles: a mechanistic approach[END_REF]. Growth room temperature was maintained at 25±2°C and 16/8 h (light/dark) photoperiod having 40 μmol/m 2 /s light intensity for all cultures was adjusted.

Establishment of callus cultures

For establishing callus, stem explant was excised from 4 week-old plantlets (in vitro). Explant was inoculated using the optimized protocol [START_REF] Anjum | Trends in accumulation of pharmacologically important antioxidant-secondary metabolites in callus cultures of Linum usitatissimum L, Plant Cell, Tissue and Organ Culture[END_REF]. Stem explants (~1.0 cm long) were inoculated on MS media modified with NAA (α-naphthalene acetic acid) 1.0 mg/L, sucrose 30g/L, agar 8g/L and pH was adjusted to 5.6 using 2 M NaOH (sodium hydroxide) prior to autoclaving (121°C, 20 min)and kept at above said conditions for growth.. The explant derived calli were subcultured after 4 weeks to ensure 100% callogenesis. The experiment was executed in triplicates. Chemicals used in experimentation were all purchased from Sigma Corporation (USA).

Cell suspension culture

For cell suspension culture, the 3-weeks old calli (subcultured) were inoculated in Erlenmeyer flasks (250 ml) containing liquid MS media with sucrose (30g/L) and 1.0 mg/L NAA. Flasks were kept on gyratory shaker (25 ± 2°C) at constant agitation (120 rpm) in 16/8 h (light/dark) photoperiod with light intensity of 40 μm/m 2 /s for 15 days. For inoculum preparation 250 ml Erlenmeyer flasks containing 100 ml Media (each flask) were used.

Elicitor preparation and treatment

Chitosan (C6H11NO4)n (deacetylating grade: 70-85%) was used as an elicitor. Chitosan was dissolved in 0.1% acetic acid at 50°C with continuous stirring for 5 hours. For elicitation liquid MS media was prepared having sucrose 30g/L and NAA 1.0 mg/L. To that media different concentrations (0.1, 0.5, 1.0, 10, 20, 50, 100, 200 and 500 mg/L) of chitosan were added. Prior to autoclaving (121°C, 20 min), pH was set to 5.6. Experiment was performed in Erlenmeyer flasks (100 ml) containing 40 ml of media. To each flask 10 ml of inoculum (15 days old fine cells) was added. Triplicate flasks were used for each concentration. Cultures were kept on gyratory shaker (25±2 °C) at constant agitation (120 rpm) in 16/8 h (light/dark) photoperiod. MS media containing sucrose 30g/L and NAA 1.0 mg/L without any elicitor was used as control.

Observation of the experiment and tracking data of the growth dynamics and secondary metabolites accumulation was executed with the gap of 5 days interval for 50 days.

Biomass determination

To determine the fresh weight (FW), respective cell cultures were harvested through filtration using 0.45 μm stainless steel sieve (Sigma), to remove any attached media, cell cultures were gently washed using dH2O. To remove the excess water, cells were gently rubbed on filter paper sheets and cells were weighed for FW. To obtain dry weight (DW) cell were dried in oven (45°C, 48 hours).

RNA extraction

Total RNA was extracted from 100 mg of liquid nitrogen ground frozen tissues using the Plant GeneJet RNA Purification Kit (Life Technologies) according to supplier instructions. Quantification of RNA was each performed using a fluorometer, with the Quant-iT RNA Assay Kit (Invitrogen) adapted for the Qubit fluorometer, according to the manufacturer's protocol.

RT-qPCR analysis

Reverse transcription was performed using a First-strand cDNA synthesis kit (Thermo).

Quantitative PCR was performed in 96-well plates with a PikoReal real time PCR system (ThermoFisher) using the DyNAmoColorFlash SYBR Green qPCR Kit (ThermoFisher). Each reaction was made in 10 μL (1 μM of each primer pairs, 0.5 μL diluted cDNAs and 2 × SYBR Green mix). All PCR reactions were carried out with the following protocol: 7 min at 95 °C, 40 cycles of 10 s at 95 °C, 10 s at 55 °C and 30 s at 72 °C. The specificity of the amplified product was confirmed for each primer pair, via a melting curve. Data analysis was carried out with Pikoreal software. Three biological replicates and two technical repetitions were performed for each sample. Relative transcript levels were obtained using specific primers (Table S1), designed with Primer3 software , and normalized via the comparative ΔΔCq method using two validated housekeeping reference genes selected by Huis et al. 24 : LuCYC encoding for cyclophilin and LuETIF5A encoding for Eukaryotic Translation Initiation Factor 5A. Results were presented on a heat map format using the MeV software computed with a hierarchical clustering analysis (HCA) representation employing the Euclidean distance as a clustering method with a complete linkage clustering as parameters.

PAL activity

Soluble proteins were extracted from 1.5 g of fresh frozen tissue by homogenization in 3 ml of 0.1 M sodium borate buffer (SBB) pH 8.8 containing 10 mM β-mercaptoethanol and kept on ice for 30 min. After centrifugation (10 min, 16,000 g) at 4°C the supernatant was collected and used in the assay. Protein concentrations were quantified using a Qubit fluorimeter and the Quant-iT Protein Assay Kit (Invitrogen) according to the manufacturer's protocol.

PAL specific activity was assayed spectrophotometrically by monitoring the production of transcinnamate at 290 nm as previously described [START_REF] Dunn | Changes in arginine, pal activity, and nematode behavior in salinity-stressed citrus in honour of Professor GH Neil Towers 75th birthday[END_REF]. The reaction mixtures contained 50 lg proteins and 50 mM l-phenylalanine in 5 ml of SBB (pH 8.8) and were incubated at 40°C.

CCR and CAD activities

Soluble proteins were extracted by grinding 1.5 g of fresh frozen tissue in a pre-chilled mortar containing a cold extraction buffer (0.1 M Tris-HCl pH 7.5, 5% w:v ethylene glycol, 2% w:v polyvinyl polypyrrolidone and 0.1 M β-mercaptoethanol). The crude extract was centrifuged (10 min, 16,000 g) at 4°C and the supernatant was used in the assays. Protein concentrations were quantified using a Qubit fluorimeter and the Quant-iT Protein Assay Kit (Invitrogen) according to the manufacturer's protocol.

CCR specific activity was determined spectrophotometrically as described previously [START_REF] Goffner | Purification and characterization of cinnamoyl-coenzyme A: NADP oxidoreductase in Eucalyptus gunnii[END_REF] using feruloyl-CoA as substrate. Feruloyl-CoA was synthesized according to the optimized protocol reported previously [START_REF] Beuerle | Enzymatic synthesis and purification of aromatic coenzyme A esters[END_REF].

CAD specific activities were determined spectrophotometrically as described earlier [START_REF] Hawkins | Purification and characterization of cinnamyl alcohol dehydrogenase isoforms from the periderm of Eucalyptus gunnii Hook[END_REF]

Plant extracts preparation

Extracts of cell cultures were prepared following the protocol described previously [START_REF] Ali | Production of commercially important secondary metabolites and antioxidant activity in cell suspension cultures of Artemisia absinthium L[END_REF] with slight modifications. Dried cell cultures were grounded finely and mixed with methanol in 1:5 (100 mg in 500 ul). All mixtures were sonicated for 30 minutes and followed by vortexing for 5 minutes, this process is repeated twice. These mixtures were then kept on gyratory shaker (25 ± 2°C) at constant agitation (80 rpm) for 24 hours. The sonication and vortexing were repeated and finally the mixtures were centrifuged at 10,000 rpm for 10 minutes and supernatant was collected and subsequently syringe filtered and stored at 4°C to be analyzed. For the extraction of lignans and neolignans from cell cultures of flax the protocol described by [START_REF] Anjum | Effects of photoperiod regimes and ultraviolet-C radiations on biosynthesis of industrially important lignans and neolignans in cell cultures of Linum usitatissimum L.(Flax)[END_REF] was followed. Extraction of lyophilized cells was carried out using 80% v/v (20 ml) methanol (aqueous) using ultra sonication (USC1200TH) having 30 kHz frequency for 1 hour at 25 ± 2 °C . The mixture was subjected to centrifugation. The supernatant was extracted and evaporated (40 °C) and followed by suspending (4 h at 40°C) in 0.1 M (1 ml) 4.8 pH buffer (citrate-phosphate). In order to release the aglycones the buffer was equipped with β-glucosidase from almonds (5 units/ml; Sigma Adrich).

Determination of total phenolic production

Total phenolic content (TPC) was evaluated according to the previously reported protocol [START_REF] Abbasi | Differential effects of in vitro cultures of Linum usitatissimum L.(Flax) on biosynthesis, stability, antibacterial and antileishmanial activities of zinc oxide nanoparticles: a mechanistic approach[END_REF] using Folin-Ciocalteu (FC) reagent. Twenty microliter of sample extract and 90 μl of FC reagent were mixed and diluted 10x using dH2O followed by incubation for 5 minutes at 25 ± 2 °C. Sodium carbonate (6%, w/v) was added to the mixture. Absorbance was recorded by Absorbance Microplate Reader (ELx808 BioTek, USA) at 725 nm. In order to plot the calibration curve (R²=0.967), 0-40 μg/ml of gallic acid used as standard. TPC was expressed as equivalents of gallic acid (GAE)/g of dry weight). Equation (I) was used to calculate the total phenolic production (TPP).

Total phenolic production = × _____________ (I)

TPP was expressed in mg gallic acid/l.

Quantification of lignans and neolignans by RP-HPLC

The extract was centrifuged for another time and prior to injection the resultant supernatant was filtered (0.

Method validation

The quantification was performed with standard calibration curves obtained using five standard dilutions ranging from 50 to 1000 μg/ml. Each standard solution was injected in triplicate.

Arithmetic means of each triplicate were calculated. The linear regression equations were carried out by plotting the peak areas against the injected amounts of standard compounds. The linearity was demonstrated by coefficient of determination (R 2 ). The limits of detection (LOD) and the limits of quantification (LOQ) were determined based on the signal-to-noise ratio (S:N) of approximately 3:1 and 10:1, respectively. Accuracy was evaluated by measuring recovery rates.

Dried flax cell cultures were homogenized and separated into two parts of equal mass, one of which was spiked with a known volume of stock solutions. The spiked and non-spiked parts were analyzed by HPLC in triplicate following the procedures described. The recovery rates were calculated according to the following formula:

Recovery rate = (amount in spiked partamount in non-spiked part) / (spiked amount) x 100.

The method precision and stability were evaluated by determining the intraday and interday variations respectively, which were calculated from data obtained by the repeated injections of standard solutions. The intraday variation was obtained by five replicates in a day and the interday variation was determined by three injections over three continuous days. Retention times and peak areas were assessed. The precision was further checked by measuring the repeatability using five continuous injections of the same extracted sample. The precision was expressed as the relative standard deviation (RSD, %).

Determination of antioxidant activity

For evaluation of antioxidant activity 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay (FRSA) was performed according to the reported protocol [START_REF] Fazal | Sucrose induced osmotic stress and photoperiod regimes enhanced the biomass and production of antioxidant secondary metabolites in shake-flask suspension cultures of Prunella vulgaris L, Plant Cell, Tissue and Organ Culture[END_REF] with minor alterations. Twenty microliter of sample extract mixed with 180 μl of DPPH (3.2 mg/100 ml methanol) and the resultant mixture was incubated at 25 ± 2°C for 60 minutes and followed by the addition of 160 μl of dH2O. Absorbance was recorded by Absorbance Microplate Reader (ELx808 BioTek, USA) at 517 nm. In order to plot the calibration curve (R²=0.989) methanolic extract of DPPH solution (0.5 ml) was used as standard. The following equation (III) was used to calculate the free radical scavenging activity as percentage of discoloration of DPPH.

Free radical scavenging activity (%) = 100 × 1 -____________ (III)

where AC stands for absorbance of the solution when sample extract was mixed at a specific concentration, and AS denotes the absorbance of standard (DPPH solution).

Ferric reducing antioxidant power (FRAP) was measured as described by [START_REF] Benzie | The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay[END_REF]

Statistical analysis

All the experiments were carried out in a synchronized manner and repeated twice. Each

Results and Discussion

Influence of different concentrations of chitosan on biomass accumulation and (neo) lignans biosynthesis

The effects of different concentrations of chitosan (0.1-500 mg/L) were evaluated on cell cultures of L. usitatissimum for biomass accumulation (FW and DW). Data recorded for chitosan-treated (each concentration) and control cell cultures after 30 days. All treatments were also analyzed for its total phenolic content (TPC). Maximum biomass accumulation (both fresh weight (FW) and dry weight (DW)) occurred at 10 mg/l of chitosan (hereafter called CHI-10) as compared to control. Morphological changes induced by different concentrations of chitosan (0.1-500 mg/L) treatments of flax cell suspensions are presented in Fig. 2A. The cell culture treated with 10 mg/l chitosan also presented the highest accumulation of TPC (Table 1) evidencing an activation of phenylpropanoid metabolism.

Following this first evaluation, to assess the impact of chitosan on phenylpropanoid pathway, transcript accumulation of monolignols as well as lignans and neolignans (i.e., monolignolderived products) biosynthetic genes by RT-qPCR (Fig. 3) and enzyme activity of key enzymes involved in monolignols biosynthesis (Fig. 4) in flax cell suspensions treated with 0 (control, CTL), 1 (CHI-1), 10 (CHI-10) and 100 (CHI-100) mg/l chitosan were evaluated.

The expression of i) monolignols biosynthetic genes LuPAL encoding for a phenylalanine ammonia lyase, LuCCR encoding for a cinnamoyl-CoA reductase and LuCAD encoding for a cinnamyl alcohol dehydrogenase, previously characterized in flax cell suspension challenging with elicitors [START_REF] Hano | Pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds[END_REF], ii) lignans biosynthetic genes, encoding for biochemically characterized enzymes, LuDIR5 encoding for a (-)-pinoresinol forming dirigent protein [START_REF] Dalisay | Dirigent protein-mediated lignan and cyanogenic glucoside formation in flax seed: integrated omics and MALDI mass spectrometry imaging[END_REF][START_REF] Corbin | A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation[END_REF], LuPLR1

encoding for a (-)-pinoresinol/(-)-lariciresinol reductase [START_REF] Hano | Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures[END_REF][START_REF] Heimendahl | Pinoresinol-lariciresinol reductases with different stereospecificity from Linum album and Linum usitatissimum[END_REF] and LuUGT74S1 encoding for a (+)-secoisolariciresinol uridine glycosyltransferase [START_REF] Ghose | Identification and functional characterization of a flax UDPglycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG)[END_REF], and iii) neolignans biosynthetic genes

LuPCBER encoding for a phenylcoumaran benzylic ether reductase [START_REF] Hano | Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures[END_REF] (Fig. 3A), were monitored after 8h, 24h and 48h after chitosan addition and their relative gene expression levels (relative to control cell suspension) are presented in Fig. 3B. Here, chitosan treatment triggered the expression of monolignols, lignans and neolignans biosynthetic genes. This gene expression stimulation appeared to be dependent on the chitosan concentration applied with a highest stimulatory effect observed with 10 mg/l chitosan treatment (CHI-10, Fig. 3B). The gene expression kinetic also appeared specific to the metabolic steps thus highlighting the possibility of a coordinated regulation of these metabolic pathways.

PAL, CCR and CAD specific activities were then monitored in control and chitosan-treated cell suspensions during a 48h period following chitosan addition (Fig. 4). In a good agreement with the gene expression analyses, a rapid, strong and dose-dependent stimulation of PAL activity was observed in all treated cell cultures, reaching a maximum activation 24h after chitosan addition (Fig. 4A). In the same way, a very similar rapid, transient and dose-dependent activation of both CCR and CAD activities were observed for chitosan-treated cells (Fig. 4B,C). From these enzyme activities data, it appeared that saturation occurred beyond 10 mg/l chitosan addition.

Activation of genes, at both expression and enzyme levels, during the early steps of the phenylpropanoid pathway in response to biotic or abiotic stresses has already been reported in other plant systems. The PAL enzyme catalyzes the entry point of L-phenylalanine into the phenylpropanoid pathway. This enzyme is known to play a crucial role in plant defense mechanisms and is presumably responsible for the increased carbon flux into this pathway leading to an increased biosynthesis of defense/stress-related compounds deriving from this phenylpropanoid pathway [START_REF] Dixon | Stress-induced phenylpropanoid metabolism[END_REF]. Chitosan induces the enzymatic defensive mechanisms in plants by producing chitinases, pectinases and glucanases enzymes and stimulates plant's immunity which results in enhanced accumulation of biomass while some reports suggest that chitosan is also responsible for enhancing the availability and uptake of water as well as essential nutrients by regulating the osmotic pressure of cells [START_REF] Bautista-Baños | Effects of chitosan and plant extracts on growth of Colletotrichum gloeosporioides, anthracnose levels and quality of papaya fruit[END_REF][START_REF] Hien | Radiation degradation of chitosan and some biological effects[END_REF]. In agreement with our results Mathew & Sankar (2012) [START_REF] Mathew | Effect of methyl jasmonate and chitosan on growth characteristics of Ocimum basilicum L., Ocimum sanctum L. and Ocimum gratissimum L. cell suspension cultures[END_REF] also reported up to 3.5 times enhancement in biomass accumulation in chitosan treated cell cultures of three species of Ocimum i.e. O. basilicum, O. sanctum and O.

gratissimum. Similar results were also reported by [START_REF] Kim | Effect of chitosan on the biological properties of sweet basil (Ocimum basilicum L.)[END_REF] for O. basilicum after application of chitosan. From its polycationic β-1,4 linked D-glucosamine polymer chemical structure, chitosan could be consider either as a biotic elicitor or fertilizer as a source of nitrogen and sugars [START_REF] Hirano | Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens[END_REF].

Here, gene expression analyses and enzyme kinetics, confirmed the trend observed for TPC measured in the corresponding cell suspensions (Table 1). From all these results, 10 mg/L concentration of chitosan was selected for further investigation.

Effect of chitosan on biosynthesis and productivity of lignans and neolignans

For determination of trends in growth kinetics and biomass accumulation data was monitored periodically at an interval of 5 days for a total of 50 days (Fig. S1). The growth curves of cell cultures of flax grown in 10 mg/L of chitosan exhibited nearly same degree of lag phase (10 days), log phase (exponential phase) starting from day 10 to day 30 comprised of 20 days and followed by 10 days of stationary phase from day 30 to day 40. From day 40 onwards decline phase was observed (Fig. 2B, Fig. S1).

Chitosan exhibited a positive influence on accumulation of total phenolic content (Fig. S2). Upon analysis it was noted that highest TPC accumulation of 19.36 mg/g DW and a productivity of 315.89 mg/l were recorded at 30 th day of inoculation. The probable explanation behind this enhancement is that chitosan upon contact with plant cells induces antioxidant defense mechanism, as a result plant synthesize phenolic compounds to scavenge harmful reactive oxygen species (ROS) [START_REF] Bahabadi | The effect of chitosan and chitin oligomers on gene expression and lignans production in Linum album cell culture[END_REF].Chitosan also has a possible key role in signaling pathways of biological synthesis of phenolics [START_REF] Dixon | Early events in the activation of plant defense responses[END_REF]. The accumulated TPC in Flax cell cultures grown in 10 mg/L of chitosan is 2.1 times (19.36 mg/g DW) higher than control (9.1 mg/g DW) (Fig 5).

Similar findings were reported previously [START_REF] Baque | Elicitor effect of chitosan and pectin on the biosynthesis of anthraquinones, phenolics and flavonoids in adventitious root suspension cultures of'Morinda citrifolia'(L.)[END_REF] who obtained a 2 fold increase in phenolic content in chitosan elicited adventitious root cultures of Morinda citrifolia.

Lignans and neolignans are polyphenols formed via phenylpropanoid metabolic pathway from a common precursor (the monolignol coniferyl alcohol). These polyphenolic compounds are pharmacologically important [START_REF] Schmidt | Lignans in seeds of Linum species[END_REF].

To get further insight in their accumulation profile and kinetics, the main lignans (SDG and LDG) and neolignans (DCG and GGCG) accumulated in flax cell cultures were quantified using RP-HPLC method (Figure S3). For this purpose the HPLC-DAD method used to quantify these lignans and neolignans, in the different plant materials analyzed in the present study, was validated in term of precision, accuracy, stability and repeatability. Results of this validation are presented in Table S2. was assessed with standard addition at three concentration levels (50%, 100% and 150%) and a good recovery of the compounds ranging from 97.5% (DCG) to 102.5 (GGCG) was observed.

In current study, lignans (SDG and LDG) and neolignans (DCG and GGCG) were accumulated in their glycosylated forms since aglycones were not detected before β-glucosidase hydrolysis (data not shown). The maximum accumulation of lignans and neolignans were observed at day 30 and 35, respectively (Figure 5). All these current results are in agreement with previously reported results [START_REF] Anjum | Effects of photoperiod regimes and ultraviolet-C radiations on biosynthesis of industrially important lignans and neolignans in cell cultures of Linum usitatissimum L.(Flax)[END_REF][START_REF] Benhamou | Induction of systemic resistance to Fusarium crown and root rot in tomato plants by seed treatment with chitosan[END_REF][START_REF] Cuero | Aflatoxin control in preharvest maize: effects of chitosan and two microbial agents[END_REF] Upon evaluation of growth dynamics it was noted that the maximum accumulation of SDG Furthermore, the maximum accumulation of dehydrodiconiferyl alcohol glucoside (DCG) was observed at 30 th day of inoculation in flax cell cultures (Fig 5C). Surprisingly, chitosan showed inhibitory effects on the accumulation of guaiacylglycerol-β-coniferyl alcohol ether glucoside (GGCG) (Fig. 5D). This observation will deserve future study in order to understand the partition regulation of the monolignol into these different monolignol-derived products upon chitosan treatment.

LDG and DCG showed growth-associated behavior, maximum accumulation observed with enhanced biomass. However, highest accumulation of SDG in late stationery phase showed that its biosynthesis was growth non-associated and its involvement if ameliorating stresses induced by nutrient depletion. Similar enhancing effects of chitosan on accumulation of lignans and neolignans have been reported [START_REF] Bahabadi | The effect of chitosan and chitin oligomers on gene expression and lignans production in Linum album cell culture[END_REF].

From these data, discrepancies can be pointed in our results from the expression levels (Fig. 3), enzyme activities (Fig. 4) and lignans and neolignans production (Fig. 5). First, the expression levels shown in Fig. 3 suggested a very rapid response of gene expression to chitosan induction after 8-24h of chitosan treatment. On the contrary, the lignans and neolignans production reached the highest levels after 35 days, whereas the expression of related genes appeared to slightly decrease after 48 h of treatment with the 10 mg/L chitosan treatment. This first discrepancy between the accumulations of lignans and neolignans concentration and their biosynthetic gene expression could result from the regulation of key genes involved in monolignol biosynthesis, as it has been reported for the effects of ABA and GA on lignin accumulation by Kim et al. [START_REF] Kim | Transcriptional regulation of the cinnamyl alcohol dehydrogenase gene from sweetpotato in response to plant developmental stage and environmental stress[END_REF] and Zhao and Dixon [START_REF] Zhao | Transcriptional networks for lignin biosynthesis: more complex than we thought[END_REF] as well as for the accumulations of lignans and neolignans [START_REF] Corbin | Identification and characterization of cis-acting elements involved in the regulation of ABA-and/or GA-mediated LuPLR1 gene expression and lignan biosynthesis in flax (Linum usitatissimum L.) cell cultures[END_REF]. For example, CAD and/or COMT genes could be the target of such regulation [START_REF] Kim | Transcriptional regulation of the cinnamyl alcohol dehydrogenase gene from sweetpotato in response to plant developmental stage and environmental stress[END_REF][START_REF] Mohr | Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes, in Arabidopsis infected with Pseudomonas syringae pv. tomato[END_REF] thus modifying the availability of the precursor monomers for lignin or lignans and neolignans biosynthesis. For instance, CAD is of particular interest, since here the CCR and CAD enzyme activities, could also appear not consistent with their corresponding gene expression levels. Indeed, the CCR and CAD enzyme activities reached their maxima 8h post-treatment and then decreased, whereas their expression remained at very high levels. Nevertheless, we have to keep in mind that CCR and CAD both belong to small multigene families, therefore the enzyme activities measured can result from the action of distinct enzymes encoded by different genes involved in different biosynthetic pathways. Here, we have determined gene expression profile of CCR and CAD genes previously associated with the production of lignans and neolignans in flax cell suspensions [START_REF] Hano | Differential accumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures[END_REF]. However, other CCR and CAD genes could be associated with the production of lignin as reported by Le Roy et al. [START_REF] Roy | Spatial regulation of monolignol biosynthesis and laccase genes control developmental and stress-related lignin in flax[END_REF] and can contribute to the overall enzyme activities measured. The induction of gene expression associated with a decrease in enzyme activity could not only be due to a differential transcriptional regulation within a multigene family, but also result from posttranslational regulation events also known to occur in the phenylpropanoid pathway. This have been, for example, described by Allwood et al. [START_REF] Allwood | Phosphorylation of phenylalanine ammonia-lyase: evidence for a novel protein kinase and identification of the phosphorylated residue[END_REF] evidencing the implication of phosphorylation event(s) and the involvement of a protein kinase in the posttranslational regulatory mechanism of the PAL enzyme. Moreover, to date no data about the turnover of proteins involved in production of lignans and neolignans are available.

Last, these apparent discrepancies between gene expression levels, enzyme activities and lignans and neolignans production might be due to the fact that the first results provide an indication of the gene expression of the enzyme activity at a precise time whereas metabolite accumulation summarize the effect during the whole duration of the experiment.

Finally, we cannot exclude a possible metabolization of chitosan into a more or less active elicitor compound(s) by flax cells following its addition to the culture medium.

Impact of chitosan-treatment on antioxidant potential of flax cell culture compared to flax seeds

DPPH assay was conducted to examine the antioxidant potential of the flax cell cultures. It was noted that the cell cultures exhibited highest antioxidant potential (93.6%) at its 30 th day of inoculation as compared to control cell cultures (Fig. 6). ROS are normally formed during light dependent processes in plants and can lead to oxidative stress which can be extremely harmful for photosynthetic cells and can damage cellular compounds like membrane lipids, proteins or nucleic acid [START_REF] Foyer | Photooxidative stress in plants[END_REF]. Chitosan and its derivatives are involved in accumulation of hydrogen peroxide (H2O2) in plants which has a key role in oxidative burst and stimulation of scavenging system of ROS in plants [START_REF] Agrawal | Chitosan activates defense/stress response (s) in the leaves of Oryza sativa seedlings[END_REF][START_REF] Lin | Hydrogen peroxide mediates defence responses induced by chitosans of different molecular weights in rice[END_REF]. Chitosan is considered to be responsible for alleviation of membrane lipid peroxidation which results in lowering the phytotoxicity in plant cells by reducing the high chemical oxygen demand. All such antioxidative activities contribute additively to the free radical scavenging capacity of a medicinal plant, therefore, free radical scavenging activity was performed in order to analyze and compare the antioxidant potential of chitosan elicited and control cell cultures of Flax. Results showed that 10 mg/L chitosan efficiently enhanced the free radical scavenging (93.6 %) compared to control (69 %). Similar results were observed for chitosan treated cell cultures of Ocimum basilicum [START_REF] Mathew | Effect of methyl jasmonate and chitosan on growth characteristics of Ocimum basilicum L., Ocimum sanctum L. and Ocimum gratissimum L. cell suspension cultures[END_REF].

Last but not least, Table 2 presents the comparison of the accumulation of lignans and neolignans and resulting antioxidant activity of the extracts prepared from control and chitosan-treated flax cell suspensions vs wild seeds (from Barbara cultivars known to be highly productive in term of lignans accumulation) [START_REF] Corbin | A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation[END_REF]; and LuPLR1 gene silenced transgenic flax [START_REF] Renouard | RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: consequences on lignans and neolignans accumulation[END_REF]. Seeds of wild type flax mainly accumulated SDG whereas seeds of transgenic RNA silencing PLR1 transgenic flax mainly accumulated neolignans. On the contrary flax cell suspensions accumulated both lignans and neolignans, and chitosan treatment was able to enhanced lignans and neolignans accumulation in this in vitro system. Moreover, flax seeds are cultivated only once a year whereas flax cell suspension constituted a continuous and efficient production system. Last important point, our in vitro production system is non transgenic and therefore could be less controversial in term of public acceptance, safety and usefulness. Altogether these results clearly indicated that flax cell suspension and in particular chitosan treatment are effective production systems of antioxidant lignans and neolignans.

Conclusion

In current study, we evaluated the impact of a number of concentrations from 0.1 to 500 mg/L of chitosan in order to elicit the biosynthesis of pharmaceutically important polyphenols i.e. lignans (SDG and LDG) and neolignans (DCG and GGCG) in L. usitatissimumcell cultures. Among all, 10 mg/l chitosan proved to be highly effective in the stimulation of gene expressions, enzyme activities and metabolite accumulation of anticancer lignans and neolignans than control cells.

Beside, chitosan (10 mg/L) also remarkably enhanced the accumulation of biomass (FW and DW) and antioxidant potential of the flax cells. The productivity of lignans and neolignans as well as antioxidant potential of these chitosan-treated cells were higher than those of flax Table S2: Validation parameters of the HPLC-DAD method for the quantification of the main lignans and neolignans from flax cell suspension extract

  treatment was consisted of triplicates. All the mean values were analyzed using Pareto analysis of variance (ANOVA). The significance at P<0.05 was determined by Duncan's multiple range test (DMRT, Windows version 7.5.1, SPSS Inc., Chicago) Graphs were generated using Origin pro(8.5).

  Representation of the peak area and standard concentrations revealed high linear correlations in the range of 50-1000 µg/mL. The linear regression of the 5-point calibration graph showed a R 2 -value ranging from 0.9989 for LDG to 0.9998 for DCG, whereas the slope of the standards covering the analytical range varied at most 1% relative standard deviation (RSD) over a four weeks period. In term of limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10), values obtained were 3.4 ng and 10.6 ng for LDG, 2.7 ng and 9.2 ng for SDG, 3.1 ng and 10.0 ng for DCG, and 4.2 ng and 13.4 ng for GGCG, respectively.The determination of the instrumental precision was realized by five injections of the same sample. The chromatographic method used proved its precision (intraday precision) with RSD values ranging from 0.45 (SDG) to 1.25 (LDG). The same sample was injected six times (0, 6, 12, 24, 48 and 72 h after its preparation) in order to evaluate the method stability (interday precision). The small observed values for the RSD, from 0.84 (SDG) to 1.65 (GGCG) confirmed the good stability of the extracted sample. Application of the whole extraction procedure three times to the same batch of material allowed the verification of the high repeatability with RSD values ranging from 0.99 (SDG) to 2.16 (LDG). The separation method accuracy measurement

occurred at 35

 35 th day (stationary phase) of inoculation (Fig 5A). Results revealed that maximum accumulation of lariciresinol diglucoside (LDG) was observed at 40 th day of inoculation in cell cultures of L. usitatissimum (Fig 5B). The accumulation of LDG in late stationary phase is showing its involvement in alleviating nutrients depletion-induced stress.

Figure 1 .

 1 Figure 1. Schematic representation of biosynthetic pathways of lignans and neolignans, quantified in cell cultures of flax using RP-HPLC-DAD adopted from Anjum et al., 4 DIR: dirigent protein; PLR: pinoresinol lariciresinol reductase; UGT/UDP-GT: UDP-Glucosyl Transferase.

Figure 2 .

 2 Figure 2. A. Morphological characteristic of flax cell cultures grown in 0.1-500 mg/L of chitosan at day 30 of culture. B. Morphological characteristic of the different growth phases of flax cell cultures grown in 10 mg/L of chitosan. B1: lag phase. B2: exponential phase. (Log) B3: stationary phase. B4: death phase.

Figure 3 .

 3 Figure 3. A. Monolignol and monolignol-derived biosynthetic pathway occuring in flax cell suspension (from Hano et al., 2006a). PAL: phenylalanine ammonia-lyase; CCR: cinnamoyl-CoA reductase; CAD: cinnamyl alcohol dehydrogenase; DIR5: dirigent protein 5; PLR1: pinoresinol lariciresinol reductase 1; UGT74S1: secoisolariciresinol uridine glucosyl transferase; PCBER: phenylcoumaran benzylic ether reductase; L-Phe: L-phenylalanine; SDG: secoisolariciresinol diglucoside; LDG: lariciresinol diglucoisde; DCG: dehydrodiconiferyl glucoside; GGCG: guaiacylglycerol-β-coniferyl ether diglucoside. B. Normalized relative gene expression profiles in flax cell suspension as determined by RT-qPCR (normalized with cyclophilin (CYC1) and Eukaryotic Translation Initiation Factor 5A (ETIF5A)) following exogenous addition of chitosan at 1 (CHI-1), 10 (CHI-10) and 100 (CHIU-100) mg/l. Results are presented as a clustering classification realized by a hierarchical clustering analysis (HCA) based on the complete linkage Pearson uncentered correlation method performed with MeV.

Figure 4 .

 4 Figure 4. Time course of specific PAL (A), CCR (B) and CAD (C) activities of the soluble protein fraction of control and chitosan-treated (at 1 (CHI-1), 10 (CHI-10) and 100 (CHIU-100) mg/l) cell suspension cultures. CTL is control cells. Bars represent SE of 3 independent experiments.

  ); a maximum accumulation for control and chitosan-treated flax cell suspension; b expressed as percentage of radical scavenging activity measured at day 30 for control and chitosan-treated flax cell suspensions; c expressed in mM of Trolox C equivalent antioxidant capacity (TEAC) measured at day 30 for control and chitosan-treated flax cell suspensions; Data are expressed as the mean of n = 4 independent experiments ± standard deviation of the mean and different superscript majuscule letters indicate significant differences between conditions (P<0.05).

Figure 3

 3 Figure 3:

Figure 5 :

 5 Figure 5:

Figure 6 :

 6 Figure 6:

  45 μm). The quantification of Lignans and neolignans aglycones was carried out using RP-HPLC by Varian liquid chromatographic system equipped with a Varian Prostar 230 pump, a

	Lignans and neolignans productivity	=	× accumulation	_________(II)

Metachem Degasit, a Varian Prostar 410 autosampler and a Varian Prostar 335 Photodiode Array Detector (PAD) and controlled by Galaxie version 1.9.3.2 software. For separation the method described by

[START_REF] Renouard | RNAi-mediated pinoresinol lariciresinol reductase gene silencing in flax (Linum usitatissimum L.) seed coat: consequences on lignans and neolignans accumulation[END_REF]

was followed using Purospher RP-18 (Merck) column (250 × 4.0 mm i.d.; 5 μm). Calibration curves were used to perform the quantification of Lignans and neolignans. In order to compare the results with published literature easily, results were expressed as mg of glycosides like SDG, LDG, DCG and GGCG equivalent per gram of dry weight. Molecular weight of compounds was used for conversion. Equation (II) was used to calculate the productivity of lignans and neolignans:

  with some modifications. Briefly, 10 μl of the extracted sample was mixed with 190 μl of FRAP (10mM TPTZ; 20mM FeCl3 6H2O and 300mM acetate buffer pH3.6; ratio 1:1:10 (v/v/v)). Incubation lasted 15min at room temperature. Absorbance of the reaction mixture was measured at 630nm with a BioTek ELX800 Absorbance Microplate Reader. Assays were made in triplicate and antioxidant capacity was expressed as Trolox C equivalent antioxidant capacity (TAEC).

Table 1 :

 1 Growth (fresh weights (FW) and dry weights (DW)) and total phenolic concentrations (TPC) in control and chitosan-treated (at different concentration levels) flax

	cell cultures
	Treatment	FW	DW	TPC
	(mg/l)	(g/l)	(g/l)	(mg/g DW)
	Control	60.35 ± 3.02	5.50 ± 0.07	9.1 ± 0.1
	0.1	187.08 ± 5.16	12.55 ± 0.15	10.37 ± 0.13
	0.5	130.58 ± 4.39	9.71 ± 0.09	13.9 ± 0.26
	1	176.08 ± 4.89	13.10 ± 0.27	14.32 ± 0.29
	10	390.66 ± 8.11	16.31 ± 0.33	19.36 ± 0.39
	20	193.25 ± 5.05	10.24 ± 0.28	15.34 ± 0.33
	100	121.66 ± 3.23	11.32 ± 0.11	13.76 ± 0.27
	200	56.33 ± 2.71	5.50 ± 0.08	10.9 ± 0.14
	500	35.83 ±1.19	12.55 ± 0.17	8.1 ± 0.09

Table 2 .

 2 Comparison of lignans and neolignans accumulations and antioxidant activities in flax cell culture (control and chitosan-treated) vs seeds (wild type seeds and RNAi-LuPLR1 transgenic seeds).

	in vitro cell cultures	seeds
			Wild type	RNAi-LuPLR1
	Control	Chitosan-treated
			(cv Barbara)	transgenic seeds
	SDG a
	4.73 ± 0.22 B	23.15 ± 2.04 A	20.62 ± 4.71 A	0.62 ± 0.15 C
	(mg/g DW)
	LDG a
	8.91 ± 0.57 B	18.26 ± 1.07 A	ND D	Traces C
	(mg/g DW)
	DCG a
	17.52 ± 0.84 B	56.25 ± 6.35 A	ND C	14.22 ± 3.67 B
	(mg/g DW)
	GGCG a
	5.52 ± 0.28 A	1.42 ± 0.67 B	ND D	Traces C
	(mg/g DW)
	DPPH
	68.36 ± 5.12 BC	93.37 ± 8.93 A	78.25 ± 11.42 B	52.14 ± 10.26 C
	(%RSA) b
	FRAP
	287.63 ± 5.38 C	485.28 ± 12.14 A	319.34 ± 6.57 B	156.45 ± 4.12 D
	(TEAC) c

ND: not detected below the LOD; Traces: detected below LOQ (see Table

2

Acknowledgements

Dr. Bilal Haider Abbasi acknowledges research fellowship of Le Studium-Institute for Advanced Studies, Loire Valley, Orléans, France. This research was in part supported by Cosmetosciences, a global training and research program dedicated to the cosmetic industry. Located in the heart of the cosmetic valley, this program led by University of Orléans, France is funded by the Region Centre-Val de Loire.

Authors Contributions

WA performed experiments of cell culture, elicitation with chitosan and analyses of TPC, TFC and antioxidant activity. AZ and MN assisted WA with all of these experiments. BHA conceived idea, supervised research and critically evaluated MS. CH contributed to HPLC analysis, performed gene expression analysis and enzymatic measurements and contributed to the conception, writing and critical reading of the manuscript. LG and SD performed HPLC. SR and JD contributed to enzymatic measurements and HPLC quantification. NGG discussed research and critically reviewed the manuscript.

Conflict of interest

Authors declare no conflict of interest

Supplementary

Seeds Plantlets Callus

Cell Suspension


R Scarpa

E Studer

S Kudriakov
email: sergey.kudriakov@cea.fr

N Chaumeix
email: chaumeix@cnrs-orleans.fr

INFLUENCE OF INITIAL PRESSURE ON HYDROGEN/AIR FLAME ACCELERATION DURING SEVERE ACCIDENT IN NPP

Flame acceleration (FA) and explosion of hydrogen/air mixtures remain key issues for severe accident management in nuclear power plants. Empirical criteria were developed in the early 2000s by Dorofeev and colleagues, providing effective tools to discern possible FA or DDT (Deflagration-to-Detonation Transition) scenarios. A large experimental database, composed mainly of middle-scale experiments in obstacle-laden ducts at atmospheric pressure condition, has been used to validate these criteria. However, during a severe accident, the high release rate of steam and non-condensable gases into the containment can result in pressure increase up to 5 bar abs. In the present work, the influence of the unburnt gas initial pressure on flame propagation mechanisms was experimentally investigated. Premixed hydrogen/air mixtures with hydrogen concentration close to 11% and 15% were considered. From the literature, we know that these flames are supposed to accelerate up to Chapman-Jouguet deflagration velocity in long obstacle-laden tubes at initial atmospheric conditions. Varying the pressure in the fresh gas in the range 0.6-4 bar, no effects on the flame acceleration phase were observed. However, as the initial pressure was increased, we observed a decrease in the flame velocity close to the end of the tube. The pressure increase due to the combustion reaction was found to be proportional to the initial pressure according to adiabatic isochoric complete combustion. p Pressure [bar] PP Piezoelectric pressure sensor

NOMENCLATURE

In case of severe accident, in-core metal-water reactions due to fuel heat-up lead to massive release of hydrogen into the containment of Light Water Reactors, as recently recalled by Fukushima accident. The safety features currently implemented for severe accident management do not avoid the possible formation of large flammable clouds. If ignited, hydrogen/air flames may accelerate up to supersonic speeds over a wide range of hydrogen concentration. Such explosions could eventually jeopardize the integrity of the containment and damage components important to safety or accident management. The structural damage depends on the maximum pressure but also on the pressure impulse [START_REF]Protection of piping systems subject to fires and explosions[END_REF]. Therefore, they are more important when the propagation velocity of the reactive wave increases.

The empirical flame acceleration criteria developed in the early 2000s by Dorofeev and colleagues [START_REF] Breitung | State-of-the-art report on flame acceleration and deflagration-to-detonation transition in nuclear safety[END_REF], [START_REF] Dorofeev | Evaluation of limits for effective flame acceleration in hydrogen mixtures[END_REF] provide effective tools for the analysis of the possible scenarios and it allows the selection of the most relevant situations for which flame acceleration (FA) may take place. Nevertheless, most of FA and DDT (Deflagration-to-Detonation Transition) criteria are based on experimental databases in which explosions were carried out at atmospheric conditions. In case of a severe accident, the high release rate of steam and non-condensable gases into the containment may result in a pressure increase up to 5 bar abs. Therefore, hydrogen explosions are more likely to happen at an initial pressure greater than the atmospheric one.

According to the thermal theory of Mallard and Le Chatelier, the laminar burning velocity is expected to vary in line with expression (1):

2 2 - ∝ n L p S , (1)
where

) ( p n n =
is the overall reaction order. For lean mixtures at atmospheric conditions 1 ≤ n , therefore L S decreases with increasing pressure. This variation is linked to the fact that the ignition temperature tends to increase as the initial pressure increases. According to [START_REF] Law | Propagation, structure, and limit phenomena of laminar flames at elevated pressures[END_REF], n strongly decreases with pressure and for lean mixtures it may reach negative values. In Fig. 1 the variation of the laminar burning velocity with pressure for 11 and 15 %vol H 2 -air mixture are shown. COSILAB® v.3 code [START_REF] Rotexo | COmbution SImulation LABoratory[END_REF], coupled with the detailed kinetic mechanism described in [START_REF] Connaire | A Comprehensive Modeling Study of Hydrogen Oxidation[END_REF], was used to compute the laminar flame speed. In the literature, we found four references dealing with the effect of initial pressure on the laminar flame velocity of a hydrogen/air mixture at room temperature [START_REF] Kuznetsov | Flammability limits and laminar flame speed of hydrogen-air mixtures at sub-atmospheric pressures[END_REF], [START_REF] Aung | Effects of pressure and nitrogen dilution on Flame/Stretch interactions of laminar premixed H2/O2/N2 Flames[END_REF], [START_REF] Kitagawa | Turbulent burning velocity of hydrogen-air premixed propagating flames at elevated pressures[END_REF] and [START_REF] Egolfopoulos | An experimental and computational study of the burning rates of ultra-lean to moderately-rich H2/O2/N2 laminar flames with pressure variations[END_REF]. At 11 vol%, the calculations underestimate the experimental results by a factor of about 3 at 1 bar. Nevertheless, the trends are the same as when the pressure increases the velocity decreases. These deviations can be explained by the difficulty of experimentally overcoming the intrinsic instabilities of the flame or by a weak kinetic scheme for these low flame temperatures. At 15 vol%, this underestimation is reduced to a factor of 2. However, the results of Kitagawa et al correspond well to the simulation and those of Aung et al show a singular behavior for pressures above twice the atmospheric pressure. At this hydrogen content, the flames are less unstable. For lean flames below equivalent ratio of 0.8, it is impossible to get rid of flame wrinkling and now it is often proposed to validate kinetic models above and then extrapolate. Nevertheless, if we look at the data collected by [START_REF] Krejci | Laminar Flame Speed and Ignition Delay Time Data for the Kinetic Modeling of Hydrogen and Syngas Fuel Blends[END_REF] for hydrogen/air mixtures under ambient temperature and pressure conditions, values between 25 and 35 cm/s for 15 vol% and between 5 and 15 cm/s for 11 vol% were obtained. Our computed results are consistent with these data. For lean mixture at 10 < p bar the activation energy increases as 5 . 0 p . Since the adiabatic flame temperature is essentially independent of pressure in the range of pressure variation considered in the present study, the Zeldovich number β is supposed to vary in the same way as the activation energy.

Moreover, by assuming that ad b T , remains constant, we make the hypothesis that the speed of sound in the burnt gas is not affected by pressure variations.

In the present work, the influence of 0 p on premixed hydrogen/air flame propagation has been experimentally investigated in the SSEXHY facility (Structures Submitted to an EXplosion of HYdrogen). The device (Fig. 2) consists of a stainless steel tube equipped with annular obstacles uniformly distributed all along the tube length. Experiments were performed at initial pressure in the range 0.6-4 bar abs. For this campaign, we chose to test mixtures with hydrogen content close to 11.0 %vol. From literature [START_REF] Kuznetsov | Effect of obstacle geometry on behaviour of turbulent flames[END_REF] we know that these mixtures are supposed to accelerate inside an obstacleladen tube up to an asymptotic velocity. This saturation velocity corresponds to the Chapman-Jouguet deflagration speed [START_REF] Chue | Chapman-Jouguet deflagrations[END_REF]. By decreasing the hydrogen concentration, the mixture is shown to be not sufficiently energetic to sustain the above mentioned condition. As a result, these flames may be partially or totally quenched depending on the obstacle blockage ratio. At atmospheric initial conditions, mixtures with 11.0 %vol hydrogen identify the lower limit for FA [START_REF] Dorofeev | Evaluation of limits for effective flame acceleration in hydrogen mixtures[END_REF]. In consequence, their behavior is quite unstable: even the slightest variation of a fundamental parameter may lead to significant modification of flame behavior.

EXPERIMENTAL SETUP

The experimental facility (Fig. 2 The acceleration tube is designed for a nominal operating pressure of 100 bar, making the device suitable for studying both deflagration and detonation mechanisms. Gas injection and venting lines are located at the flanged heads. The first head also hosts in its centre an automotive spark plug used to ignite the flammable mixture, as shown in Fig. 3.

Thirty-two equally spaced annular obstacles have been installed inside the tube with the aim of promoting turbulence at the wall. Obstacle blockage ratio (

2 D d = ) / ( 1 BR -
) is equal to 0.3. Three thin threaded rods form the structure supporting the obstacles. The stainless steel annular obstacles (5 mm thick) present three holes, 120° spaced from each other, for rods insertion. Obstacles are 120 mm spaced from each other, resulting in a 125 mm uniform pitch. Air (20.1% O 2 -70.9% N 2 ) and hydrogen are consecutively injected into the tube from two separated gas tanks. Prior to gas injection, lines are vented by removing the residual gas with a vacuum pump, thus avoiding any contact between hydrogen and oxygen. This precaution allows us to achieve a flammable mixture only inside the tube. At the end of the injection process, the injection system is inerted with nitrogen. Hydrogen concentration can be varied inside flame propagation limits, covering the whole spectrum of possible mixtures from lean mixtures to rich ones, including stoichiometric conditions. Once the initial conditions of the experiment are established, i.e. pressure and hydrogen concentration, the method of partial pressures is used to prepare the desired mixture inside the tube. Three pressure sensors are available for three different pressure ranges: a vacuum gauge in the range 0-133 mbar, a pressure transmitter in the range 0-1000 mbar, while the last one covers the range 0-5 bar.

Since hydrogen and air are injected at different times, a concentration gradient is formed along the tube axis. An ATEX gas recirculation pump then promotes mixture homogenisation. The mixture is forced to recirculate into an external loop for about 30 minutes before reaching the homogeneous condition. This period of time was calibrated from pre-tests using helium instead of hydrogen and measuring the local concentration with thermal conductivity gauges. Close to the recirculation loop entrance at the tube wall, two taps are available for gas sampling. Gas samples are then analysed by a gas chromatograph (Agilent 490 µGC). The uncertainty in gas composition is in the order of 0.1 %vol.

In the 3-modules configuration, the combustion tube is equipped with 40 instrumentation ports. The instrumentation configuration for the present experimental campaign involved fourteen photomultiplier tubes (PMT), nine piezoelectric pressure sensors (PP) and fourteen piezoelectric shock sensors (CC). Sensors disposition along the tube is detailed in Tab. 1. Here the origin 0 = x corresponds to the inner surface of the first flanged connection, located 5 mm away from the origin of the fluid domain, as shown in Fig. 3. A cross sectional view of the tube shows the position of the sensors on the tube diameter (A -A section in Fig. 3). On the vertical position (on the top generatrix of the tube) we can find dynamic pressure sensors (Kistler 601A, 6001 and 7001 types). For the 601A and 6001 types we have used a flush mounting nut, while 7001 type are recessed mounted. At 90° from the pressure sensors, we can find the optical access for the Hamamatsu R11568 photomultiplier tubes (PMT). These detectors collect the UV light emitted by OH • radicals located at the reaction front within a very narrow solid angle (the uncertainty on the flame position for the collimated beam is 66 . 3 max = x δ mm). As the flame tip passes through the PMT solid angle, a negative voltage is recorded. This signal allows us to extrapolate the time-of-arrival of the flame tip, monitoring flame propagation along the tube axis.

On the other side, Chimiemetal piezoelectric sensors are located. These detectors allow the detection of a shock wave in the unburnt gas. Thanks to their small sensitive area (2 mm diameter), they can be used to extrapolate the velocity of the pressure wave ahead of the flame in a more accurate way. These sensors are not calibrated and strongly affected by the radiation emitted by the flame. As a result, once the flame passes by the sensor, the piezoelectric crystal respond with a strong current pulse. Their use is therefore restricted to the detection of the shock waves ahead the flame, in the unburnt gas.

Tube inner wall, as well as obstacles surface, was thermally treated with black oxides to prevent light reflection. The accuracy of optical measurements is therefore increased.

National Instrument NI PXIe-1078 data acquisition station was used to record sensors output signals.

The five data acquisition cards 12-bit, 8-channels NI PXIe-5105 (with onboard memory) allow a maximum record frequency of 60 MHz per card. Acquisition trigger and signal record are controlled via LabVIEW 2015 platform. The program is coded to launch signal acquisition as the ignition spark is initiated.

RESULTS AND DISCUSSION

Test conditions are presented in Tab. 2. A repeatability study was performed at 1 0 = p bar in order to compare the results obtained in the present work with those from literature. In Fig. 4(a) the velocity of the flame tip along the tube axis is presented for the test cases 3, 4 and 5 ( % 11 2 H ≈ χ ). Present results were compared to those from [START_REF] Kuznetsov | Effect of obstacle geometry on behaviour of turbulent flames[END_REF] for the same mixture composition and obstacle blockage ratio.

In Fig. 4(a) the error band associated to velocity measurement technique is also presented for the three test cases. This uncertainty is related to the error in the flame position measurement introduced by PMT solid angle. In Fig. 4 The experiments performed with a more reactive mixture match perfectly the data presented in [START_REF] Kuznetsov | Effect of obstacle geometry on behaviour of turbulent flames[END_REF], while for mixtures with 11% hydrogen data dispersion is wider. These flames show in fact a quite unstable behaviour that reflects in the repeatability study. As shown in Fig. 4(a), the experimental device is not long enough to allow the flame to reach the CJ deflagration velocity for a 11 %vol hydrogen mixture.

Sensors output signals were recorded at 100 kHz. To eliminate the noise related to electronics and structures resonance, a low-pass filter was applied to the signals. The cut-off frequency was chosen to preserve burnt gas acoustics among the obstacles. The frequency of an acoustic wave propagating in the burnt gas inside the chamber delimited by two obstacles can be computed as follows: m/s after the fourth one and so on. The shock wave is therefore less energetic at each step. Decaying speed of sound 701, 655, 630, 594 m/s correspond to the average temperature for combustion products 1220, 1060, 980, 875K, respectively, decaying due to energy losses. As the initial pressure increased, the leading shock wave can no longer be considered mono-dimensional. As a result, pressure signals are characterized by a multitude of peaks with the same frequency (corresponding to a sound wave travelling along the tube) but different phase (see Fig. 7(a) and Fig. 8(a)).

D c f sb a 2 = . ( 2
The influence of the initial pressure on the flame velocity is shown in Fig. 6. For the leanest mixture, test cases were sorted in two groups depending on the hydrogen concentration. In Fig. 6(a For 11% in air, in the first stage of flame acceleration no significant differences with pressure variation were observed. Measurement spread is in fact comparable to that one shown in the repeatability study. Thus, even if the laminar burning velocity decreases of a factor 9 from 0.6 bar to 4 bar, as shown in Fig. 1, it seems to have little influence on the first phase of flame acceleration. However, the burning rate ρ u S L (Fig. 6(d)) is indicative of the rate of consumption of the fresh gases and it decreases slightly when the pressure increases. There are therefore other parameters relevant for the flame acceleration mechanism that intervene in compensating the decrease in burning rate. Moreover, these flames are thermally unstable. Since the Lewis number is an invariant of pressure, Le~ const~0.347, the product β(Le-1) is always negative and lower than the threshold stability value -2.

In Fig. 6(c) the effect of initial pressure on flame velocity for hydrogen/air mixture with 15 vol% is presented. For p 0 = 0.6 and 1 bar, the velocity profiles are perfectly superposed up to x/D=15 despite a large variation of the burning rate. For p 0 greater than 2 bar, instead, a greater acceleration rate is observed from the early stages of flame acceleration. As pointed out in Fig. 6(c) the burning rate continues to rise and at the same time the product β(Le-1) becomes even more negative. It seems that it is mainly the initial phase, which is faster, and therefore this acceleration can be mainly attributed to the variations of the thermodiffusive parameters. The σ -criterion described in [3] is satisfied for all the cases considered in this work. This criterion identifies a necessary condition for flame acceleration for a given mixture and geometry. According to [START_REF] Kuznetsov | Effect of obstacle geometry on behaviour of turbulent flames[END_REF], at large scale the threshold value * σ depends only on the mixture composition and the initial conditions. This limit is defined by the condition 100 / ≥ δ T L , where T L is the integral mixing length and δ is the flame thickness. The expansion ratio, σ , does not depend on pressure and, for a mixture with 11 %vol hydrogen, its value coincide with the threshold value for FA found by Dorofeev and colleagues at atmospheric condition, = * σ 3.7. Nevertheless, in our experiments, by increasing the initial pressure, the ratio δ / T L decreases, as shown in Fig. 7. Here, the definitions given in [START_REF] Dorofeev | Evaluation of limits for effective flame acceleration in hydrogen mixtures[END_REF] were used to compute the integral mixing length and the flame thickness:

D L T = (3) L b S T σ α δ ) ( = , (4)
where ) ( b T α is the thermal diffusivity in the burnt gas. For 100 / < δ T L , the geometry plays an important role in the flame propagation mechanism. By increasing the turbulence of the gas flow ahead of the flame, flame stretching may then cause flame slowdown or extinction. As shown in Fig. 6, the last stage of flame propagation (starting approximately with the 20 th obstacle) is characterized by a decrease of flame velocity. This slowdown is more pronounced as the initial pressure increases. Anyway, global flame quenching did not occur. At the end of the tube, the flame front is also perturbed by the reflected pressure wave. As the reflected shock interacts with the flame, hydrodynamic forces tend to slowdown the flame. Fig. 8 and Fig. 9 show the pressure variations recorded by PP6 and PP9 for different tests. In these pipe sections (Fig 3 right), a PMT is associated with a pressure sensor. In order to compare the pressure measurements, curves are shifted in time based on PMT time-response at the correspondent pressure sensor position. Flame tip time-of-arrival is also presented in the graphs with a vertical black line. From the graphs, we can state that the pressure increase due to the combustion reaction is directly proportional to the initial pressure according to adiabatic isochoric complete combustion pressure (AICC). For completeness, the values of the adiabatic isochoric combustion pressure AICC Finally, in Fig. 10 the pressure evolution recorded for test case 5 by the sensors located at the end of the tube is shown. We can see that shock sensors response is coherent with pressure sensors one. The pressure increase at the end of the tube is also consistent with an adiabatic isochoric complete combustion, as above mentioned. Additionally, the propagation of a reflected shock wave moving at the speed of sound in the burnt gas is traced with a purple line. In its way back, this wave further compresses the burnt gas. As a consequence, OH • radicals population in the exited state increases. The photomultiplier tubes capture the light emitted during the transition to the ground state. As a result, in Fig. 10 PMT signals are characterized by the presence of an additional peak, of significantly lower amplitude if compared to the first one.

CONCLUSIONS

The effect of initial pressure on lean hydrogen/air flame acceleration was experimentally investigated. The initial pressure was varied in the rage 0.6-4 bar abs. In the early stages of flame acceleration, no significant changes were observed except for 15 vol% at 2 bar. In the central module, where the velocity increase is almost constant, a wide data dispersion was noticed for the leanest mixture. However, this spread is similar to that one encountered in the repeatability study. Therefore, even if the laminar burning velocity decreases of almost one order of magnitude from 0.6 bar to 4 bar, as shown in Fig. 1, it seems to have little influence on the first phase of flame acceleration.

As the initial pressure was increased, we observed a decrease in the flame velocity close to the end of the tube. The reason of this flame slowdown is mainly due to the interaction between the reflected shock wave and the flame. Fig. 7(b) and Fig. 8(b) show that the pressure increase due to the combustion reaction is directly proportional to the initial pressure. Therefore, the hydrodynamic force generated by the shock and acting on the flame becomes stronger, slowing down the flame in its propagation along the tube.

Further experimental campaigns are planned to complete the study. For these new experiments, we envisage to investigate the influence of the initial pressure on different mixture compositions, including rich mixtures. Obstacle size variation is also foreseen. In future works, the effect of the dynamic pressure peaks on simple stainless steel structures, such as plates and cylinders, placed at the end of the accelerating tube will also be analysed. A visualization module with quartz windows will be also available to perform optical flame front tracking measurements.

Present and future studies provide a solid base of experimental data for the validation of CFD models devoted to the simulation of LWR severe accident scenarios.

Figure 1 .

 1 Figure 1. Laminar burning velocity vs. pressure: (a) 11 %vol H 2 -air mixture (φ = 0.296), (b) 15 %vol H 2 -air mixture (φ = 0.422).

  and Fig. 3) is an obstacle-laden tube designed to study the acceleration mechanisms of premixed hydrogen/air flames. The tube is composed of 4 modules of the same length 1310 = l mm and internal diameter 120 = D mm. In the present experimental campaign only three modules are exploited for a total length of 3930 mm. If this study had focused on the chocked flame regime it would have been more appropriate to use four sections. As the acceleration profile was the goal of this work, the three sections provided enough information.

Figure 2 .

 2 Figure 2. SSEXHY facility in the 3-modules configuration.

Figure 3 .

 3 Figure 3. Schematic of the experimental device. Lengths are given in mm.

  Here the error band represents minimum and maximum values over three repeated shots.

Figure 4 .

 4 Figure 4. Repeatability study at atmospheric pressure ( 1000 0 = p mbar). Experiments performed during the present work are compared to those from [7] for the same mixture composition and obstacle blockage ratio.

5 ,

 5 cut-off frequency for the filtering process was then imposed to be a c f f ≥ . To avoid damping the amplitude of those harmonics close to a f signals from Test 1 are presented in a 70 ms time window. Since the acquisition is triggered by the current that supplies the electrical spark, the exact time 0 t at which the ignition of the mixture takes place is unknown. In the graph, signals are plotted according to their non-dimensional position D x / along the tube. The solid large black lines identify tube modules borders. Both recorded pressure signals (light grey lines) and filtered ones (red lines) are presented in Fig.5. Signals recorded by piezoelectric sensors correspond to the dynamic overpressure. The absolute pressure can then be obtained by adding to the overpressure the initial value of the pressure. An important thing to point out is that the radiation emitted by the flame strongly influences the response of those pressure sensors in flush mounting position (PP1, PP3 and PP4). By comparing PP2 and PP3 signals, we can note that the sensor in the recessed mounting position undergoes a lower thermal load.The time-of-arrival diagram of the flame tip, drawn from photomultiplier tubes response, is also presented in Fig.5with a blue solid curve. For the sake of clarity, shock sensor output signals are not presented in the graph.The saw-tooth shape of the pressure signals visible in Fig.5is caused by the shock waves reflecting on the head flanges. For the test cases with 1000 0 ≤ p mbar, a quasi-mono-dimensional leading shock is formed. Post-processing pressure signals from Test 1, we found that, after the first reflection at the end of the tube, the shock wave propagates at almost 701 m/s. At each reflection, its velocity decreases:

Figure 5 Figure 6 .

 56 Figure 5. Test 1: 600 0 = p mbar and

Figure 7 Figure 8 .Figure 9 .

 789 Figure 7.

  in Fig 8(b) and Fig. 9(b).

Figure 10

 10 Figure 10. Test 5: 1003 0 = p mbar and

Table 1 .

 1 Instrumentation configuration.

	Location [mm]	Shock sensors	Pressure sensors (type)	Photomultiplier tubes
	265			PMT1
	460	CC1	PP1 (6001)	PMT2
	655	CC2	PP2 (7001)	PMT3
	850	CC3	PP3 (6001)	PMT4
	1045	CC4	PP4 (601A)	PMT5
	1575	CC5	PP5 (7001)	PMT6
	1770	CC6		PMT7
	1965	CC7	PP6 (7001)	PMT8
	2160	CC8		PMT9
	2355	CC9	PP7 (7001)	PMT10
	2885	CC10		PMT11
	3080	CC11	PP8 (7001)	PMT12

Table 2 .

 2 Test matrix.

	Test #	0 p [mbar]	2 χ [%] H	Test #	0 p [mbar]	2 χ [%] H
	1	600	10.9	11	600	14.8
	2	800	11.1	12	600	14.4
	3	1000	11.1	13	1000	14.6
	4	1000	11.2	14	1000	14.6
	5	1003	10.9	15	1000	14.9
	6	1501	11.1	16	1001	15.0
	7	1502	10.9	17	1002	14.8

ACKNOWLEDGMENTS

The work was performed at the French Alternative Energies and Atomic Energy Commission (CEA). Authors thank Air Liquide and EDF for supporting current works.