Nibras Belgaroui

Ikram Zaidi

Ameny Farhat

Hichem Chouayekh

Nadia Bouain

Sandrine Chay

Catherine Curie

Stephane Mari

Khaled Masmoudi

Jean-Claude Davidian

Prof M Hanin

Stéphanemari

Pierre Berthomieu

Hatem Rouached
email: hatem.rouached@supagro.inra.fr

Moez Hanin
email: moez.hanin@cbs.rnrt.tn

Over-expression of the Bacterial Phytase US417 in Arabidopsis Reduces the Concentration of Phytic Acid and Reveals Its Involvement in the Regulation of Sulfate and Phosphate Homeostasis and Signaling

Keywords: Homeostasis , phosphorus, phytic acid, phytase, sulfate, signalling Plant Pi, inorganic phosphate, MS, Murashige and Skoog, P, phosphorus, PA, phytic Homeostasis, phosphorus, phytic acid, phytase, sulfate, signalling

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

F Introduction

Phosphorus (P) is an essential element for optimal plant growth and animal nutrition. Plants serve as the major entry point for this element into the food chain. P is absorbed by plant roots from the soil predominantly as inorganic phosphate (Pi), which is present in very low concentrations [START_REF] Marschner | Mineral nutrition of higher plants[END_REF]Poirier andBucher, 2002, Lin et al. 2014). At the end of the plant life cycle, Pi is mainly stored in the seed as phytic acid (PA), which is a myo-inositol ester with six phosphate groups [START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF]. The abundance of PA in crop seeds used as food and feed is a long-standing issue. PA is considered as a key anti-nutrient element for humans and non-ruminant animals [START_REF] Raboy | Seed phosphorus and the development of low-phytate crops[END_REF][START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF]. Indeed, the PA-associated P is in general not bioavailable to the non-ruminant animals because they produce little or no phytase in their digestive tract [START_REF] Nys | Occurrence of phytase in plants, animals, and microorganisms[END_REF]Wodzinski et al. 1996). Phytases are special acid phosphatases with the capacity to hydrolyze PA by cleaving the Pi off the inositol backbone [START_REF] Lei | Phytase, a New Life for an ″Old″Enzyme[END_REF]Yao et al., 2011). They can be classified into four families namely histidine acid phosphatases, β-propeller phytases, purple acid phosphatases and more recently protein tyrosine phosphatase-like phytases (for review see [START_REF] Lei | Phytase, a New Life for an ″Old″Enzyme[END_REF]). Thus, monogastric diets need to be supplemented with Pi to meet the P requirement for animal growth. PA has additional anti-nutritional properties since it acts as a chelating agent for several essential cations such as iron (Fe), preventing their absorption, and thus contributing to mineral deficiencies [START_REF] Maga | Phytate: Its chemistry, occurrence, food interactions, nutritional significance, and methods of analysis[END_REF][START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF][START_REF] Veum | Effect of graded levels of iron, zinc, and copper supplementation in diets with low-phytate or normal barley on growth 31 performance, bone characteristics, hematocrit volume, and zinc and copper balance of young swine1[END_REF]). On the other hand, high PA content in cereal seeds is one of the major P sources that contribute to eutrophication, which is aggravated by the release of Pi from the manure [START_REF] Bali | Microbial phytases in nutrition and combating phosphorus pollution[END_REF]. Therefore, reducing the PA levels in grain would contribute to improving animal and human diets through better micronutrient resorption, and decreasing the negative impact of P excess on the environment. The regulation of Pi homeostasis, transport and signaling in plants are highly regulated by complex processes [START_REF] Rouached | Regulation of phosphate starvation responses in plants: signaling players and cross-talks[END_REF][START_REF] Lin | Long-distance call from phosphate: systemic regulation of phosphate starvation responses[END_REF]. At the transport level, multiple Pi transporters implicated in acquiring Pi at the root surface, distributing Pi to various organelles and organs have been identified including members of the PHT1 and the PHO1 gene families [START_REF] Nussaume | Phosphate import in plants: focus on the PHT1 transporters[END_REF][START_REF] Stefanovic | Overexpression of PHO1 in Arabidopsis leaves reveals its role in mediating phosphate efflux[END_REF][START_REF] Bouain | Phosphate and zinc transport and signalling in plants: toward a better understanding of their homeostasis interaction[END_REF]. At the signaling level, some regulatory pathways describing plant response to Pi starvation have been proposed [START_REF] Bari | PHO2, microRNA399, and PHR1 define a phosphate-signaling pathway in plants[END_REF]Lin et al. 2008a;[START_REF] Rouached | Regulation of phosphate starvation responses in plants: signaling players and cross-talks[END_REF][START_REF] Lin | Long-distance call from phosphate: systemic regulation of phosphate starvation responses[END_REF], including key regulators such as the Arabidopsis noncoding RNAs AT4 and IPS1, which were first described as phosphate starvation induced genes [START_REF] Shin | Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation[END_REF]. Mutants affected in the above cited genes cause an alteration of Pi inter-organ distribution (for review [START_REF] Lin | Long-distance call from phosphate: systemic regulation of phosphate starvation responses[END_REF][START_REF] Lott | Elements present in mineral nutrient reserves in dry Arabidopsis thaliana seeds of wild type and pho1, pho2, and man1 mutants[END_REF]. The transport of Pi in plant is also influenced by a complex interconnection between the Pi homeostasis and those of essential nutrients such as iron (Fe) [START_REF] Ward | The effect of iron on the primary root elongation of Arabidopsis during phosphate deficiency[END_REF][START_REF] Bournier | Arabidopsis FERRITIN 1 (AtFer1) gene regulation by the PHOSPHATE STARVATION RESPONSE 1 (AtPHR1) transcription factor reveals a direct molecular link between iron and phosphate homeostasis[END_REF][START_REF] Bouain | Phosphate and zinc transport and signalling in plants: toward a better understanding of their homeostasis interaction[END_REF].Therefore, it is clear that future work that aims to modulate PA content in seed by manipulation of the Pi transport and/or signaling should first consider the complexity of these regulatory pathways.

In crop plants, various genetic and biotechnological strategies have been employed to reduce the PA contents [START_REF] Lucca | Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains[END_REF][START_REF] Brinch-Pedersen | Concerted action of endogenous and heterologous phytase on phytic acid degradation in seed of transgenic wheat (Triticumaestivum L.)[END_REF][START_REF] Chiera | Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability[END_REF][START_REF] Drakakaki | Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron[END_REF]. The classical mutagenesis approach has been used to generate mutants having low phytic acid (lpa) content [START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF]. This strategy was shown to be effective in cereals including wheat [START_REF] Guttieri | Identification and characterization of a low phytic acid wheat[END_REF]), maize [START_REF] Raboy | Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1[END_REF][START_REF] Pilu | Phenotypic, genetic and molecular characterization of a maize low phytic acid mutant (lpa241)[END_REF][START_REF] Shi | The maize low-phytic acid 3 encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds[END_REF], and rice [START_REF] Larson | Isolation and genetic mapping of a non-lethal rice (Oryza sativa L.) low phytic acid 1 mutation[END_REF]Kim et al. 2008a, b). The maize mutants, namely lpa1-1 and lpa2-1, show a reduction in PA content by 66% and 50% in mature seeds respectively [START_REF] Raboy | Origin and seed phenotype of maize low phytic acid 1-1 and low phytic acid 2-1[END_REF]. Molecular studies led to the identification of the genes responsible for the lpa2 and lpa3 phenotype in maize. LPA2 and LPA3 have been shown to be an inositol phosphate kinase and a myo-inositol kinase, respectively [START_REF] Shi | The maize lowphytic acid mutant lpa2 is caused by mutation in an inositol phosphate kinase gene[END_REF]; ATP-binding cassette (ABC) transporter [START_REF] Shi | Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds[END_REF]). However, many other genes responsible for the lpa phenotype remain largely unknown. It is important to note that the reduction in PA levels in the lpa mutants is sometimes associated with the alteration of agronomic traits such as crop yield (Roby, 2009). For this reason, [START_REF] Shi | Embryo-specific silencing of a transporter reduces phytic acid content of maize and soybean seeds[END_REF] proposed a new alternative where through silencing of the MRP-ABC transporter in an embryo-specific manner, they successfully produced maize and soybean seeds with low-PA and high-P contents and which germinate normally without significant decreases in seed dry weights.

However, such a strategy was not always successful since in rice the seed-specific silencing of a MRP (osMRP5) causes a reduction of PA levels but also a decrease in seed weight accompanied by reductions in seed germination and seedling emergence [START_REF] Li | Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice[END_REF].

Despite its primary importance, the biological role of PA in plants remains poorly understood either under standard growth conditions or under challenging environments. A different strategy is now actively pursued, which is the generation of transgenic crops by manipulating the PA biosynthetic pathway [START_REF] Kuwano | Molecular breeding for transgenic rice with low phytic acid phenotype through manipulating myo inositol 3 phosphate synthase gene[END_REF][START_REF] Kuwano | Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter[END_REF]Suzuki et al.2007[START_REF] Zhao | Characterization of OsMIK in a rice mutant with reduced phytate content reveals an insertion of a rearranged retrotransposon[END_REF][START_REF] Kim | Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach[END_REF]. Such a strategy becomes feasible in the light of a better understanding of the PA biosynthetic pathway, which involves both inositol-lipid intermediates and a lipid-independent pathway (Stevenson-Paulik et al.2005;[START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF].

Some genes of the PA biosynthetic pathway have been cloned such as the inositol 3phosphate synthase (MIPS) and several inositol polyphosphate kinases (IPK1, IPK2, IPK3, INK1) [START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF]. In rice, lpa lines were generated by seed-specific down regulation of MIPS, the key enzyme which catalyzes the conversion of glucose 6-phosphate into inositol monophosphate (Ins(3)P), the early substrate in PA biosynthetic pathway (Ali et al. 2013a;[START_REF] Kuwano | Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter[END_REF]). The IPK1gene is required for the later steps of PA synthesis and its [START_REF] Kim | Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach[END_REF]. Furthermore, molecular and physiological analyses of several lpa mutants revealed the relevance of IPKs in phosphorylating IP intermediates. Recently, the RNAi-mediated seed-specific silencing of OsIPK1 resulted in a substantial reduction in seed PA levels without negatively affecting the growth and development of transgenic rice plants (Ali et al. 2013b). This finding strongly supports the possibility of uncoupling low PA content from plant growth capacity.

On another hand, multiple attempts to increase Pi availability via PA hydrolysis through heterologous expression of microbial phytases in planta have been successfully made in the last decade [START_REF] Brinch-Pedersen | Engineering crop plants: getting a handle on phosphate[END_REF][START_REF] Lei | Phytase, a New Life for an ″Old″Enzyme[END_REF]. In most cases, a fungal phytase of Aspergillus niger or bacterial phytases from Bacillus subtilis strains were expressed in transgenic plants as intracellular or secreted forms [START_REF] Richardson | Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate[END_REF][START_REF] Brinch-Pedersen | Engineering crop plants: getting a handle on phosphate[END_REF][START_REF] Yip | The introduction of a phytase gene from Bacillus subtilis improved the growth performance of transgenic tobacco[END_REF][START_REF] Lung | Secretion of betapropeller phytase from tobacco and Arabidopsis roots enhances phosphorus utilisation[END_REF][START_REF] Bilyeu | Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase[END_REF]. So far, numerous transgenic plants heterologously expressing a phytase have been developed, but one ideal enzyme for all applications does not exist. Therefore, screening for phytases with suitable catalytic properties and stability is of primary interest. In this report, we explored the effects of a novel thermostable phytase PHY-US417 from B. subtilis [START_REF] Farhat | Gene cloning and characterization of a thermostablephytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme[END_REF]) on plant growth in Arabidopsis. Our results showed that the overexpression of an intracellular form of PHY-US417 leads to a significant decrease in the PA content, which is concomitant with a higher Fe remobilization during seedling establishment compared to control plants.

Results

The purified bacterial phytase PHY-US417 promotes growth of Arabidopsis seedlings under P-limited conditions Few reports described previously the contribution of extracellular phytases or externally applied phytases in promoting the plant-growth capacity under Pi stress conditions [START_REF] Idriss | Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect[END_REF][START_REF] Lung | Phytase activity in tobacco (Nicotiana tabacum) root exudates is exhibited by a purple acid phosphatase[END_REF][START_REF] Richardson | Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate[END_REF]. In this study, the β-propeller phytase PHY-US417 of Bacillus subtilis US417 was chosen because of its interesting catalytic properties since the in vitro enzymatic activity exhibited a great pH stability with an optimum at pH 7.5, and was previously reported to be specifically active on PA [START_REF] Farhat | Gene cloning and characterization of a thermostablephytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme[END_REF]. We were interested here to investigate whether these enzymatic properties can be of a beneficial effect for plant growth under P-limited conditions. For this reason, wild type Arabidopsis seedlings were germinated on standard MS growth medium and then transferred to low-P medium containing PA and in the presence or the absence of the purified enzyme PHY-US417. Compared to standard conditions, seedlings grown on low-P medium are dwarfed and exhibit small dark green leaves, which are typical Pi deficiency features (Figure 1A). When PA (0.33mM) was supplied as the sole P-source in the growth media, the growth of seedlings is also inhibited but to a less extent in comparison with those grown on low-P medium (Figure 1A). In the presence of the purified phytase PHY-US417 together with PA as a sole P-source, the seedlings grow far better, and even more vigorously than those grown on a standard MS.

Under these conditions, the purified phytase seems to stimulate significantly root growth (Figure 1B), and the seedlings display up to ~ 5 times more shoot and root fresh biomasses than those grown on low-P medium (Figure 1C). Like other β-propeller phytases, PHY-US417 is expected to hydrolyse PA by sequential removal of only 3 Pi groups to finally generate myo-inositol triphosphates [START_REF] Greiner | Pathway of phytate dephosphorylation by beta-propeller phytases of different origins[END_REF][START_REF] Kerovuo | Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism[END_REF], and the addition of PA at a concentration of 0.33 mM would result in the release of ≅ 1mM Pi, the usual concentration used in a standard MS medium. Therefore, this finding indicates that under P-limited conditions, the bacterial phytase is capable to restore and promote the growth of Arabidopsis seedlings most likely by hydrolyzing the PA present in the medium leading to the increase of the Pi bio-availability.

Generation of transgenic Arabidopsis plants overexpressing the bacterial phytase PHY-US417

The capacity of the supplied phytase PHY-US417 to promote plant growth under P-limited conditions prompted us to investigate the effect of the overexpression of the phy-US417 gene in Arabidopsis plants. The full length ORF encoding the mature form of this phytase (presumably cytoplasmic according to protein subcellular localization prediction programs, such as WoLF PSORT software http://wolfpsort.org) was first cloned in the binary vector pCAMBIA1302 under the control of the 35S promoter and the NOS terminator. The vector also bears the HPT gene (conferring resistance to hygromycin) as a selectable marker for plant transformation. After Agrobacterium-mediated transformation of Arabidopsis plants, several transformants were selected on an hygromycin-containing medium (see Materials and Methods). PCR screenings on genomic DNA of a number of T1 transformants allowed the amplification of both HPT and phy-US417 transgenes (supplemental figure 1B, C).

Homozygous T3 plants were then generated, where both transgenes were shown to be stably expressed (supplemental figure 1D) at seemingly variable levels that might be linked to the transgene integration sites. Two transgenic lines (L7 and L9) harboring single transgene inserts were finally chosen for further molecular and physiological analyses.

The transgenic lines overexpressing phy-US417 gene exhibit higher phytase activities and lower seed PA concentrations To assess whether PHY-US417 is active in planta, two week-old seedlings from both lines L7 and L9 grown on MS medium, were harvested for phytase assays. These assays were performed under optimal conditions (pH 7.5 and 55°C) for PHY-US417 but not for endogenous Arabidopsis phytases. Our results showed that phytase activities were approximately 8 and 28 times higher in the extracts of lines L7 (1.31±0,81U/g FW) and L9

(4.79±0,64U/g FW), respectively, in comparison with the wild-type control (0,17±0.04 U/g FW). Together with the expression data of the phy-US417 gene (supplemental figure 1D), this finding demonstrates that Arabidopsis plants can produce an active form of this phytase.

The effects of the phy-US417 gene overexpression on the PA accumulation were determined in the seeds of the two transgenic lines L7 and L9 and wild type plants (Col) grown under standard condition as described by [START_REF] Haug | Sensitive method for the rapid determination of phytate in cereals and cereal products[END_REF]. Our results showed that both transgenic lines contain ~ 40% less PA compared to the control (Figure 2), thus demonstrating that the phytase PHY-US417 activity in planta resulted in decreasing the PA contents.

The overexpression of phy-US417gene in Arabidopsis improves plant growth after P resupply

In order to assess the effect of the constitutive overexpression of phy-US417 on the tolerance of the transgenic lines L7 and L9 to Pi starvation, plant growth and fresh and dry masses were evaluated in plants grown for 5 to 20 days under Pi deficiency and then transferred to standard MS medium containing 1mM KH 2 PO 4 (+Pi) for 10 additional days (Figure 3). The Arabidopsis ipk1-1 mutant known to accumulate low PA level (Stevenson-Paullik et al. 2005) was also included in this experiment. Both transgenic lines show a better recovery from Pstarvation than wild-type plants (Figure 3A). Although to a less extent, the ipk1-1 mutant has also a better capacity to recover from P starvation. After ten days of growth on MS medium, L7 and L9 lines exhibited higher growth rates (especially of roots) which were illustrated by The rate of Fe remobilization is higher in low phytic acid lines compared to wild-type plants PA is deposited in seeds within protein storage vacuoles as mixed salts [START_REF] Raboy | Approaches and challenges to engineering seed phytate and total phosphorus[END_REF]) that are made with different metal cations such as Fe. In Arabidopsis, it has been shown that during germination, the Fe pool is remobilized from mature embryos to feed the developing tissues of the seedling [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF]. Here, the effect of lowering seed PA on the Fe remobilisation during germination was examined using an Fe histochemical detection assay as described previously [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF]). The Fe staining of the wild type, ipk1-1 and the two PHY-overexpressor lines reveal that in mature embryos the Fe pattern is identical in all genotypes (Figure 4A). However, two days after germination it is clear that the concentration of Fe in the vascular tissues of the mutant and transgenic lines is significantly lower than in the wild-type, indicating that during the two days of post-germinative growth, the rate of Fe remobilization was higher in the mutant lines, compared to the wild type (Figure 4B). These data strongly suggest that the decrease in PA levels, via the overexpression of the phy-US417 gene may enhance the Fe remobilization during seed germination in Arabidopsis.

The PHY-US417 overexpression alters phosphate and sulfate concentrations in

Arabidopsis.

We have investigated the effects of lowering PA levels in the PHY-overexpressing lines on the concentrations of major inorganic anions including Pi. concentrations revealed that under P-starvation (-Pi), the transgenic lines accumulated ~40-50% more Pi in their shoots than the wild type (Figure 5). Once transferred to medium with added Pi, the differences in shoot Pi-accumulation between wild type plants and transgenic lines were attenuated. In the roots, no significant differences were recorded between tested lines, neither under P starvation nor during the recovery phase. Interestingly, among other inorganic anions, the SO 4 anions accumulated to higher levels in the shoots (up to 45% increase) of the transgenic plants during Pi starvation (-Pi) and the recovery phase (+Pi), compared to wild-type plants (Figure 5).There is a less significant difference regarding SO 4 concentrations in the roots of the lines tested. No significant changes were recorded for other inorganic (i.e. nitrate) and organic anions (i.e. malate) in shoots or roots of the transgenic lines, in comparison to wild-type plants (data not shown). Therefore, these data demonstrate that the overexpression of PHY-US417 in P-limited Arabidopsis plants, affects inter-organ distribution of two essential macronutrients, Pi and SO 4 .

Expression of phosphate and sulfate transporters is up-regulated in the PHY-US417

transgenic lines.

To better understand the underlying molecular mechanisms by which the reduction of the PA levels in the PHY-overexpressors affects Pi and SO 4 inter-organ distribution, we performed expression analysis of a subset of transporters: PHT1;1, PHT1;4, PHO1, and PHO1;H1 (for Pi) (Stefanovich et al. 2007;[START_REF] Misson | Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants[END_REF][START_REF] Shin | Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low-and high-phosphate environments[END_REF]) and SULTR1;1 and SULTR1;2 (for SO4) [START_REF] Yoshimoto | Two distinct high-affinity sulfate transporters with different inducibilities mediate uptake of sulfate in Arabidopsis roots[END_REF]. This expression analysis included also the nitrate (NO3) uptake transporters, NRT1;1 and NRT2;1, [START_REF] Forde | Nitrate transporters in plants: structure, function and regulation[END_REF][START_REF] Tsay | Nitrate transporters and peptide transporters[END_REF]) as well as IRT1 gene which is involved in Fe uptake [START_REF] Vert | IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth[END_REF]. Both transgenic lines (L7 and L9), the ipk1-1 mutant and wild-type plants were grown under -Pi conditions for 15 days and then transferred to standard conditions (+Pi) for 10 days, and the mRNA accumulation levels of these root transporters were determined by quantitative RT-PCR as described by but not PHO1, show an up-regulation in both L7 and L9 transgenic lines compared to wildtype plants (Figure 6). Also, the transcript accumulation of SULTR1;2, but not SULTR1;1, increases in these lines. No significant changes in the expression levels of the two tested NO 3 transporters, and of the IRT1 gene, were observed. This result clearly indicates that the overexpression of PHY-US417, and consequently the reduction of PA levels, may affect both the Pi and SO 4 transport systems in Arabidopsis. Of note, the ipk1-1 mutant shows expression patterns similar to those observed with both transgenic lines, which strongly suggests that the PA is involved in the signalling cascades regulating the expression of a subset of genes involved in Pi and SO 4 transport in the plant.

The overexpression of the phy-US417 gene affects phosphate sensing and signalling in plants

Because the L7 and L9 transgenic lines appear to have altered Pi homeostasis (similar to the ipk1-1 mutant), and show a greater root growth capacity after Pi-starvation, we were interested to see whether the sensitivity to extracellular Pi supply was also altered. Seeds from the PHY-US417 overexpressor lines, the ipk1-1 mutant, and wild-type plants were germinated in a vertical position on low-P MS medium for 5 days and then transferred to Pi-containing MS medium (+1 mM Pi) for 5 additional days. Subsequently, light microscopy observations of root sections revealed that both transgenic lines and the ipk1-1 mutant produce long and numerous root hairs, which were completely absent on the roots of wild type plants (Figure 7A). These data, which are in line with those reported for the ipk1-1 mutant (Stevenson-Paullik et al. 2005), strongly suggest that the overexpression of this bacterial phytase reduces the capacity of Arabidopsis to sense extracellular Pi levels, since L7 and L9 transgenic lines are unable to form shorter root hairs at higher Pi concentrations. To strengthen our hypothesis, RNAs, IPS1 and AT4, in the phy-US417 transgenic lines and ipk1-1 mutant in comparison to wild type plants grown on standard media (1 mM Pi). Interestingly, our results showed that compared to wild type plants, these marker genes are both induced in the transgenic lines albeit to lower levels than in the ipk1-1mutant (Figure 7B). Therefore, this finding reinforces the idea that lowering PA levels, either in the PHY-US417 overexpressing lines or in the ipk1-1 mutant affects Pi signalling in Arabidopsis.

Discussion

A comprehensive understanding of the role of PA in plant biology is of a great scientific interest, with implications for biotechnological and agronomic strategies aimed at reducing PA levels in the grain. PA is known as the storage compound of phosphorus in seeds.

Divergent effects of lowering the PA content on seed germination potential have been reported [START_REF] Kuwano | Generation of stable 'low phytic acid' transgenic rice through antisense repression of the 1D-myo-inositol 3-phosphate synthase gene (RINO1) using the 18-kDa oleosin promoter[END_REF][START_REF] Nunes | RNAi-mediated silencing of the myo-inositol-1-phosphate synthase gene (GmMIPS1) in transgenic soybean inhibited seed development and reduced phytate content[END_REF][START_REF] Campion | Isolation and characterisation of an lpa (low phytic acid) mutant in common bean (Phaseolus vulgaris L.)[END_REF][START_REF] Doria | Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant[END_REF]Nusrat et al. 2013[START_REF] Zhao | Characterization of OsMIK in a rice mutant with reduced phytate content reveals an insertion of a rearranged retrotransposon[END_REF]. In this study, we have generated Arabidopsis transgenic PA is a natural cation chelator [START_REF] Borg | Iron transport, deposition and bioavailability in the wheat and barley grain[END_REF], because of its negatively charged Pi side groups. It is generally assumed that the appropriate release of essential ions during seed germination is crucial to determine seedling vigor. Interestingly, the PHY-US417 transgenic lines, as well as the ipk1-1 mutant, show a stronger remobilisation of Fe during germination compared to wild type (Figure 4). Therefore, it is tempting to propose that reduction of PA level leads to an increase in the proportion of bioavailable Fe in the endosperm, hence improving seedling establishment. Although the site of Fe storage is now well described in Arabidopsis embryos, the chemical form of this vacuolar pool of Fe remains unknown [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF]). Nevertheless, the co-localization of Fe and P atoms in globoids inside the storage vacuoles [START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF] has led these authors to propose that Fe may be bound to PA and thus that during germination, the remobilization of Fe may require specific biochemical processes to release Fe atoms from this type of ligand. Although not demonstrated in the present report, the increased remobilization of Fe in mutants affected in their PA content is a strong indication that in the globoids, Fe is associated to PA and that specific enzymes may be required to allow the correct solubilisation of Fe before its efflux from the vacuole [START_REF] Lanquar | Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron[END_REF]. Taken together, these results suggest that the normal germination potential and the growth promotion observed in the transgenic lines described here may be due to the phytase activity of PHY-US417, which by PA hydrolysis can reduce the harmful effects of PA linked to its binding capacity to essential cations for plant metabolism, such as Fe (Raboy et al.2009). This finding represents the first experimental illustration for a link between PA and Fe remobilisation during seedling establishment.

The regulation of plant growth is a complex process and involves several phytohormones, such as auxin, which may interact with PA. It has been reported that auxin response mutants, Interestingly, a link between PA and auxin has been established, since PA was found in crystals of the auxin receptor TIR1, and discussed as potent functional cofactor; but its precise role remains to be confirmed [START_REF] Tan | Mechanism of auxin perception by the TIR1 ubiquitin ligase[END_REF]). Similarly inositol pentakisphosphate (InsP5) was found to be a cofactor of jasmonate receptor COI [START_REF] Sheard | Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor[END_REF]. Therefore, the high affinity of PA and InsP5 with two homologous plant hormone receptors, COI1 and TIR1, offers profound implications for the role of inositol polyphosphates in plant hormone signaling. In our study, whether the alteration in PA concentration in the transgenic lines expressing the phy-US417 gene interferes with auxin and or jasmonate biosynthesis or responses (which ultimately may improve plant growth), requires further investigation.

On the other hand, regulation of ion homeostasis plays a key role in determining plant growth and responses to various environmental stresses. Results presented here provide lines of evidence that the heterologous expression of an intracellular phytase can improve Arabidopsis growth capacity under nutritional stress, namely Pi deficiency, without any apparent negative traits. The low-PA transgenic lines, L7 and L9, exhibit better growth under Pi-starvation compared to the control, as they appear more vigorous with expanded roots and shoots after stress recovery, suggesting a potential role for PA in the response of plants to Pi availability.

The majority of low-PA mutants (i.e. Atipk1-1 mutant) described in the literature exhibited an increase in inorganic phosphate, which is in favour of improving the availability of Pi and other micronutrients, such as Fe that are chelated by PA. The current study further supports this general observation (Figure 4 and Figure 5). Of note, the PHY-US417 overexpressing lines show an increase of intracellular Pi and SO4 concentrations in their shoots, under Pistarvation, indicating an apparent defect in the 'rheostat' that monitors the Pi and SO4 status of the leaves. Once transferred to standard conditions, the shoot Pi and SO4 concentrations levels remains higher in ipk1-1mutant and transgenic lines in comparison to wild type. The mutant. An interaction between SO4 and Pi homeostasis has been reported [START_REF] Rouached | The transcription factor PHR1 plays a key role in the regulation of sulfate shoot-to-root flux upon phosphate starvation in Arabidopsis[END_REF]), but no metabolite has yet been identified in higher plants that affects, at gene expression and physiological levels, the response to both elements. Our finding introduces PA as an attractive candidate for controlling Pi and SO4 transport and homeostasis. PA is known to accumulate in large amounts in seeds but much less in other organs including leaves [START_REF] Bentsink | The genetics of phytate and phosphate accumulation in seeds and leaves of Arabidopsis thaliana, using natural variation[END_REF][START_REF] Raboy | Myo-inositol-1,2,3,4,5,6-hexakisphosphate[END_REF]. It has been also shown that PA synthesis can occur in vacuoles of cell suspension cultures [START_REF] Mitsuhashi | Phytic acid synthesis and vacuolar accumulation in suspension-cultured cells of Catharanthus roseus induced by high concentration of inorganic phosphate and cations[END_REF]. In this work, we showed that the PA levels decrease in the seeds of PHY-overexpressors. Whether PA contents are also affected in other organs of these transgenic lines, remains to be determined. Although PA concentrations in mature roots/shoots were not detected by the method used, the results indicated that the modulation of PA levels may affect directly or indirectly the expression of P-related genes and root architectural traits. It is likely that such effects could be due not only to Pi mobilization but also to regulatory, and in particular hormonal networks.

In addition, it appears that low-PA transgenic lines L7 and L9 are affected in Pi sensing.

Indeed, the microscopic analysis revealed that in contrast to wild-type plants, the roots of both transgenic lines develop root hairs even when Pi was supplied in the media. These observations are in line with the previous report by [START_REF] Stevenson-Paulik | Generation of phytate-free seeds in Arabidopsis through disruption of inositol polyphosphate kinases[END_REF]. Taken together, these results strongly suggest that low PA levels interfere with the regulation of Pi sensing. The Pi signalling is also altered in these lines, since the expression of IPS1 and AT4

genes is up-regulated in the roots of the transgenic lines grown under standard conditions whereas in wild type plants, both genes are only induced under Pi-starvation [START_REF] Burleigh | The down-regulation of Mt4-like genes by phosphate fertilization occurs systemically and involves phosphate translocation to the shoots[END_REF][START_REF] Liu | Differential expression of TPS11, a phosphate starvation-induced gene in tomato[END_REF]. It is worth to note that IPS1 and AT4 genes are also known to interfere with the shoot-to-root Pi distribution [START_REF] Shin | Loss of At4 function impacts phosphate distribution between the roots and the shoots during phosphate starvation[END_REF]. In this context, given their 18 similar phenotypes, it will be interesting to explore a possible genetic interaction between Arabidopsis at4 or ips1 and the low PA lines.

In conclusion, this report demonstrates that the bacterial phytase PHY-US417 is effective in Arabidopsis and its expression leads to a significant decrease in the seed PA content, without any effect on the seed germination potential. Such a feature would encourage exploiting this bacterial phytase as a biotechnological means for sustainable and environmentally friendly agriculture. The data presented here strongly support an active role of PA in the regulation of Pi and SO 4 homeostasis including transport and signalling. The future challenge will be to develop a comprehensive understanding of the role of PA in the acquisition and assimilation of these two elements by discovering new regulatory networks. Therefore, this knowledge will be certainly valuable not only for an effective and a more targeted approach to engineering low-phytate crops, but also to fully appreciate the role of PA in the regulation of plant physiology and metabolism. For growth analyses under low phosphate conditions, 7 day-old wild-type seedlings grown on MS agar medium (Murashige and Skoog, 1962) were transferred onto the following media:

standard MS medium (with 1mM KH 2 PO 4 ) or low-P MS medium (with 5 µM KH 2 PO 4 ) in which Pi was replaced with 0.33 mM PA without or with added phytase PHY-US417 (6.5U) purified as previously described [START_REF] Farhat | Gene cloning and characterization of a thermostablephytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme[END_REF].

For recovery tests, seedlings were first grown on low-P MS medium for 5, 10, 15 or 20 days and then transferred carefully to complete MS medium for 10 days. After photo documentation, the plant materials (shoots and roots) were collected for fresh and dry weight determinations, as well as quantitative real-time PCR and High-Performance Ion Chromatography (HPIC) analyses.

For light microscopy, seedlings were grown on vertical plates on low-P medium for 5 days and then transferred to MS medium for further 5 days. Then roots were regularly scanned using an Epson Perfection 1240U scanner.

Molecular cloning and generation of transgenic plants

The nucleotide sequence encoding the mature form of the phytase PHY-US417 (accession no. AM501550) of Bacillus sutilis [START_REF] Farhat | Gene cloning and characterization of a thermostablephytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme[END_REF]) was amplified by PCR with PfuTurbo This binary vector contains also the hygromycin resistance gene (HPT) as a selection marker.

The resulting construct pC1302-phy US417 (Supplemental figure 1A) was used to transform

Arabidopsis thaliana (ecotype Columbia, Col-0) plants by floral dipping using Agrobacterium strain GV3101 (Clough and Bent, 1998).

Transgenic plants were selected on MS agar medium supplemented with 15 µg/ml hygromycin. From twenty T1 independent transformants, six T2 lines showing a 3:1 segregation for hygromycin resistance (indicative of single transgene insertion) were grown up to T3 generation to identify plants homozygous for the transgene and from which two lines, L7 and L9, were chosen for further detailed molecular and physiological analyses.

Molecular characterization of transgenic lines

Genomic DNA was prepared from fresh leaves of independent transformants. The presence of phy-US417 and HPT genes was verified by PCR screening. The forward and reverse primers for HPT gene were HPTIIF (5'ATGAAAAAGCCTGAACTCAC3') and HPTIIR (5' CTCT-ATTTCTTTGCCCTCG3'), and those for the phy-US417gene were PHYC-Bg and PHYC-Sp.

To perform expression analysis by RT-PCR, total RNA was first extracted from 200 mg of two week-old seedlings of wild type Arabidopsis and transgenic lines using Trizol reagent were reverse-transcribed using M-MLV reverse transcriptase (Invitrogen). The reverse transcription (RT) reactions were performed at 37°C for 1 h using 2 µM oligo-dT18. Two microlitres of first strand cDNAs were employed as a template for PCR amplifications with a pair of phy-US417-specific primers, PHYC-Bg/PHYC-Sp or HPT-specific primers HPTIIF/HPTIIR. An Arabidopsis Actin gene fragment, used as an internal control, was amplified with the primers: ActF: 5' GGCGATGAAGCTCAATCCAAACG-3' and ActR: 5'-GGTCACGACCAGCAAGATCAAGACG-3'. For PCR reactions, samples were denatured for 5 min at 94 °C and then run for 30 cycles of 30 sec at 94 °C, 1 min at 56°C, and 30 sec at 72 °C with a final extension of 5 min at 72 °C. Finally, the RT-PCR products were separated by agarose gel electrophoresis.

Quantitative RT-PCR

Total RNA was isolated from frozen root tissues of Arabidopsis plants using the RNeasy Plant mini Kit (Qiagen) according to the manufacturer's protocol. RNA samples were quantified with Nano Drop spectrophotometer (Thermo Scientific) and cDNA was synthesized from 1 µg of total RNA using an oligo(dT) primer and M-MLV reverse transcriptase (Promega). Quantitative real-time RT-PCR was performed with the LightCycler® 480 Real-Time PCR System (Roche) using SYBR green dye technology. PCR reactions were done in a 10 µl containing 500 nM of each forward and reverse primers, 5 ul of the SYBER green I master and 1 µl of 1:3 cDNA dilution. Reactions were performed in lightCyber® 480 Multiwell Plates 384 (Roche) covered with optical film (Sarstedt). Specific primers used for quantitative real-time RT-PCR analysis are described in [START_REF] Pfaffl | Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR[END_REF] and [START_REF] Rouached | Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis[END_REF]. All mRNA levels were calculated from threshold cycle values Ubiquitin 10 (UBQ10) transcript levels according to [START_REF] Gruber | Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection[END_REF].

Phytase Activity assay

Phytase activity was estimated colorimetrically by monitoring the release of Pi from PA (Na-InsP6; P-8810; Sigma) as previously reported [START_REF] Engelen | Simple and rapid determination of phytase activity[END_REF]. Briefly, two-week old seedlings (three replicates, 20 plants per replicate) were ground in a mortar and pestle at 4°C

with three volumes (v/w) of MES/Ca buffer (15 mM MES buffer with 0.5 mM CaCl2, pH 7.5) containing 1 mM EDTA. Extracts were then centrifuged for 10 min at 12,000g and from which100 µg were reacted at 55°C for 30 min in a 100 mM NaOAc (pH 7.5) assay buffer containing 2 mM PA as a substrate. The reaction was terminated with the addition of an equal volume of 10% (w/v) trichloroacetic acid. The released Pi was quantified spectrophotometrically at 820 nm with molybdate-blue method [START_REF] Murphy | A modified single solution method for the determination of phosphate in natural waters[END_REF].

Background readings due to Pi contamination (time 0) were subtracted before calculation.

One unit (U) of phytase is the activity that releases 1 µmol of phosphate per minute under these assay conditions. The protein concentrations were determined according to Bradford method (1976) using bovine serum albumin as a standard.

Phytic acid measurement

The seed PA contents were determined by colorimetric method as described previously [START_REF] Haug | Sensitive method for the rapid determination of phytate in cereals and cereal products[END_REF]. Four mg of seeds were extracted with 0.4 N HCl during 3.5 h with constant stirring at room temperature (RT). The extract was transferred into a glass tube with a stopper ammonium iron (III)-sulfate solution (0.02% NH 4 Fe(III)-sulfate in 0.2 N HCl).

Next, the mixture was heated at 95°C for 30 min and cooled on ice for 15 min, followed by an incubation at RT for 20 min. Then, the mix was centrifuged at 4000g at room temperature for 30 min and the supernatant was transferred to another glass tube to which 1% (w/v) 2,2'-

High-performance ion chromatography analysis

Anion content measurements were performed as described by [START_REF] Rouached | Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis[END_REF]. Briefly, after determining the mass of the fresh shoot and root materials, anions were extracted by incubating the tissue in Milli-Q water heated at 70°C for 60 min. The extract was centrifuged for 10 min at maximum speed, and the supernatants were passed through 0.2 µm filters.

Anion content of the final clear supernatant was determined by HPIC (LC20 Dionex) using an IonPaq AS11 column and a sodium hydroxide linear gradient [START_REF] Rouached | Differential regulation of the expression of two high-affinity sulfate transporters, SULTR1.1 and SULTR1.2, in Arabidopsis[END_REF]. The identification and quantification of each anion were performed using the Chromeleon software (Dionex).

Iron histochemical detection

Embryos were dissected from dry seeds after 2h of imbibition in water. Seedlings were germinated on half-strength MS medium without sucrose and Fe and grown for 2 days at 21°C in long days (16h light). For Fe histochemical detection, mature embryos and 2-day-old seedling were stained using the Perls/DAB procedure described by [START_REF] Roschzttardtz | Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis embryo[END_REF].

The presence of Fe is revealed as brown pigments.

Statistical analyses

Statistical analyses of the data were performed using analysis of variance (ANOVA) and the (A) Seedlings were first grown on MS medium for one week then transferred to standard MS medium (MS with 1 mM Pi, as a control), or to low-P medium (MS-P), in the absence or in the presence of 0.33 mM phytic acid (PA) and the phytase PHY-US417 (6.5U). (B)

Photographs of the roots of seedlings grown under the above conditions. All photographs were taken 15 days after transfer. Representative data of three biological replicates are shown.

(C) Fresh weights of shoots and roots from seedlings grown under conditions indicated in A).

Measurements were recorded 15 days after transfer. Data are mean± SD of three individual replicates. Each replicate was obtained from the analysis of shoots and roots collected from a pool of six plants. PHY-US417 transgenic lines L7 and L9. All plant genotypes including wild type Col-0, were grown in the green house and seeds were collected at the end of their life cycle. Seed PA concentrations were determined by colorimetric method as described previously (Haug and Lantzsch,1983) and expressed as µmol PA/g seed. Data are mean± SD of three individual replicates. Each replicate was obtained from the analysis of seeds collected from a pool of six plants.

F o r P e e r R e v i e w

  the maize lpa1 gene encodes a multidrug resistance-associated protein (MRP)

  up to 50%) in fresh and dry masses compared to wild-type plants (Figure3B, C). These data demonstrate that the overexpression of the bacterial phytase promotes Arabidopsis growth during recovery from Pi-starvation.

  .(2008). The transcript abundances of the PHT1;1, PHT1;4, and PHO1;H1,

  the transcript accumulation of the two Pi starvation induced non coding

  lines compromised in their ability to accumulate PA by the overexpression of an intracellular form of the B.subtilis phytase PHY-US417. Interestingly, the decrease in PA levels in PHY-US417 transgenic seeds did not alter the germination potential, and even resulted in a better seedling establishment. So far, only few studies have described normal seed germination behaviour in plants accumulating lower levels of PA. In rice, silencing of the IPK1 gene can mediate a substantial reduction in seed PA levels without negatively affecting the growth and development of transgenic plants(Ali et al. 2013). In Arabidopsis, the ipk1 mutation causes a severe decrease in PA level without altering the kinetics of seed germination or the cotyledon's appearance(Stevenson-Pauliket al. 2005). So far, the transgenic lines reported in together with the ipk1-1 mutant, offer a good opportunity to assess the biological role of PA in seed germination in Arabidopsis.

  aux1, affect plant development and particularly root growth[START_REF] Pitts | Auxin and ethylene promote root hair elongation in Arabidopsis[END_REF]).

  the expression of genes involved in Pi and SO4 transport is consistent with the accumulation of Pi and SO4 in the shoot of the PHY-overexpressors and ipk1-1

  photoperiod of light/dark cycle of 8 h/16 h, light intensity of 250 µmol•m -2 •s -1 , and

  Stratagene; La Jolla, CA, USA), using the PHYC-Bg (5'GCAGATCTATGTCCGATCCTTATCATTTTACC3'), and PHYC-Sp (5'GCACTA-GTTTATTTTCCGCTTCTGTCGGTC3') primers, which harbour the BglII and SpeI restriction sites respectively. An ATG start codon was introduced into the forward primer PHYC-Bg, immediately upstream the coding sequence of the mature peptide. The amplified fragment was first subcloned into the pGEM-T easy cloning vector (Promega) and then introduced as BglII-SpeI fragment into the pCAMBIA1302 binary vector between the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator.

  according to the manufacturer's protocol.To remove contaminating DNA, RNAs were treated with DNaseI (Promega) at 37°C for 15 min. DNase-treated RNA samples (2 ug)

  [START_REF] Livak | Analysis of relative gene expression data using realtime quantitative PCR and the 2 -∆∆ C T Method[END_REF] and as relative to controls and normalized with respect to

  (v/v) thioglycolic acid was added. After a defined time, absorbance was measured at 519 nm.

Figure 2 .

 2 Figure 2. Measurement of seed phytate concentrations in wild type plants and in the two

Figure 3 .

 3 Figure 3. Growth performance of Arabidopsis PHY-US417 overexpressing lines grown under

Figure 4 .

 4 Figure 4. Fe remobilization is faster in low phytate mutants during germination.

Figure 5 .

 5 Figure 5. Effect of the overexpression of PHY-US417 in Arabidopsis on organ phosphate and

Figure 6 .

 6 Figure 6. Effect of the overexpression of PHY-US417 in Arabidopsis on the expression of a

Figure 7 .Supplemental figure 1 .FFigure 1 .Figure 2 .FCFigure 3 .

 71123 Figure 7.The overexpression of PHY-US417 gene alters Pi sensing and signalling in

Figure 4 .-Figure 5 .Figure 6 .

 456 Figure 4. Fe remobilization is faster in low phytate mutants during germination.Fe was stained with Perls/DAB in mature embryos extracted from dry seeds (upper panels) or in 2-days old seedlings (of wild type, ipk1-1, and transgenic lines L7 and L9) after germination in liquid medium without Fe (lower panels).

  Effect of in vitro phytase supply on the growth of Arabidopsis wild type plants.

	Figures legends
	Figure1.
	F o F o F o F o F o F o F o F o F o
	r r r r r r r r r
	P P P P P P P P P
	e e r e e r e e r e e r e e r e e r e e r e e r e e r
	R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w
	Tukey's test to compare mean values. Only differences with P values < 0.05 were considered
	as significant.
	24 25

Plant & Cell Physiology

Acknowledgments

This work was supported by grants provided by the Ministry of Higher Education and Scientific Research (Tunisia) and the Institut National de la Recherche Agronomique (France). Authors would like to thank Drs Zaigham Shahzad and Sikander Chowdhary for their critical reading of this manuscript.


Anouchah Momeni

Elena M Staicu Casagrande

Alexia Dechaux

Hocine Khemliche
email: hocine.khemliche@u-psud.fr

Ultra-Fast Crystallization Dynamics at an Organic-Inorganic Interface Revealed in Real Time by Grazing Incidence Fast Atom Diffraction

The poor structural properties of organic-inorganic interfaces and their variability represent the main cause of device under-performance. Understanding and controlling the development of these properties in real time has been a difficult experimental challenge. Using a recent technique based on grazing incidence fast atom diffraction (GIFAD), we were able to directly observe during deposition structural transitions in a perylene monolayer on Ag(110). Crystallization from the liquid phase occurs into two distinct structures with drastically different dynamics. Transition to the most compact packing occurs by self-organization only after a second layer has started to build up; subsequent incorporation of molecules from second to first layer triggers an ultra-fast crystallization on a macroscopic sale. The final compact crystalline structure shows a long range order and superior stability, which opens good perspectives for producing in a controlled manner highly ordered hybrid interfaces for photovoltaics and molecular electronics.

The size, the anisotropy and the flexibility of organic molecules give rise to a complex organization dynamics when deposited on inorganic substrates. [START_REF] Kowarik | Organic molecular beam deposition: fundamentals, growth dynamics, and in situ studies[END_REF] These considerations are largely responsible for both polymorphism [START_REF] Mayer | Dynamics of bimodal growth in pentacene thin films[END_REF][START_REF] Jones | Substrate-induced and thin-film phases: polymorphism of organic materials on surfaces[END_REF] and the lack of long range order in thin organic films. For applications that require good charge transport properties, such as molecular electronics, [START_REF] Klauk | Organic thin-film transistors[END_REF] the level of organization and homogeneity are crucial properties. Because these depend strongly on the deposition process and parameters, growth monitoring in real time by surface sensitive and nondestructive techniques appears essential.

Various techniques have been used to continuously monitor the growth of thin organic films. In particular, reflectivity curves have been used with different probes, electrons in Reflection High Energy Electron Diffraction 5 or X-rays, [START_REF] Mayer | Growth dynamics of pentacene thin films: Real-time synchrotron x-ray scattering study[END_REF][START_REF] Kowarik | Real-time studies of thin film growth: Measurement and analysis of X-ray growth oscillations beyond the anti-Bragg point[END_REF][START_REF] Sparenberg | Controlling the growth mode of para-sexiphenyl (6P) on ZnO by partial fluorination[END_REF] to gain information of growth modes. However, the subsequent damage to the film, shown to being due to secondary electrons, [START_REF] Graham | Damage to trifluoroacetamido-terminated organic self-assembled monolayers (SAMs) on aluminum, titanium, copper and gold by aluminum K.alpha. x-rays is due principally to electrons[END_REF] remains a critical issue. [START_REF] Kowarik | Thin film growth studies using time-resolved x-ray scattering[END_REF] Atom scattering at grazing incidence probably represent the best alternative. It is extremely sensitive to surface quality in terms of defect density (adatoms, step edges). In the diffractive mode, [START_REF] Schuller | Diffraction of fast atomic projectiles during grazing scattering from a LiF(001) surface[END_REF][START_REF] Rousseau | Quantum scattering of fast atoms and molecules on surfaces[END_REF] it gives access at first order to the periodic profile of the valence electron density. [START_REF] Khemliche | Grazing Incidence Fast Atom Diffraction: an innovative approach to surface structure analysis[END_REF] Grazing Incidence Fast Atom Diffraction (GIFAD) [START_REF] Khemliche | Device and method of characterizing surfaces[END_REF] has been used for resolving crystallographic properties of a thin organic film. [START_REF] Seifert | Surface structure of alanine on Cu(110) studied by Fast Atom Diffraction[END_REF] Here, we show that GIFAD is also able to provide in real time (see figure S1) valuable information on the structural dynamics of an organic monolayer without any damage to the film.

Perylene deposited on Ag(110) represents a good prototype of a weakly interacting system. The good balance between intermolecular and molecule-substrate forces makes the organization mode extremely sensitive to substrate preparation and growth conditions. The main features of the monolayer formation have been well described. [START_REF] Seidel | Structural transitions of perylene and coronene on silver and gold surfaces: A molecular-beam epitaxy LEED study[END_REF] With increasing density, a gas-like phase transforms into a liquid-like phase, [START_REF] Gao | Understanding and controlling the weakly interacting interface in perylene/Ag(110)[END_REF] which further evolves into a crystalline monolayer under various structures. Besides these general observations, the gas-to-liquid and liquid-to-solid phase transitions have not been accessible and the characteristics of the final monolayer structure show large discrepancies. [START_REF] Gao | Understanding and controlling the weakly interacting interface in perylene/Ag(110)[END_REF][START_REF] Dou | Aggregation of organic molecules on silver surface with the balance between molecule-substrate interaction and intermolecular interaction[END_REF] These facts call for a better understanding of the phase transitions, which might only be accessible by monitoring continuously the growth process by a surface sensitive and non-destructive tool.

In grazing incidence atom scattering, topographic defects translate directly into a loss of reflectivity. For this reason, follow up of this parameter during thin film growth provides stringent clues of the growth process in addition to providing endpoint detection. For instance, GIFAD could effectively monitor the layer-by-layer homoepitaxial growth of GaAs(100). [START_REF] Atkinson | Dynamic grazing incidence fast atom diffraction during molecular beam epitaxial growth of GaAs[END_REF] Figure 1a shows the time evolution of the reflectivity for 500 eV He atoms during perylene deposition on Ag(110). The beam is aligned along the [1-10] direction of the substrate. The reflectivity curve is obtained by summing the intensity inside a rectangular zone having a width of 1.8° and 1.5° along the polar and azimuthal directions respectively. Opening the evaporator shutter produces a drop of the reflectivity, which reaches a minimum at 320 s. At this stage, molecules can be seen as low density randomly distributed protrusions that obstruct specular scattering of the probe beam until roughly a coverage of half a monolayer. Above this coverage, it is the voids between molecules that become defects; thus the reflectivity increases as the voids are being progressively filled. A peak is eventually reached and, as deposition proceeds, the reflectivity decreases until a weak but sharp revival followed by an abrupt drop. Then the evaporator shutter is closed and the reflectivity slowly recovers. It is noteworthy that the shutter is closed at a precise moment determined by prior experiments. As shown in figure S3, when deposition proceeds further, the curve exhibits a significant change of slope and falls continuously.

The height of the peak at 1800 sec. points to a very smooth monolayer that perfectly wets the substrate. The remarkable feature lies in the weak reflectivity gain followed by a sharp drop around 2500 s, such an anomaly could be the signature of an unusual phase transition. Note that the slope of decrease is much steeper than observed at start of deposition where molecules adsorb parallel to the surface plane. [START_REF] Gao | Understanding and controlling the weakly interacting interface in perylene/Ag(110)[END_REF] One can notice the asymmetry of the reflectivity curve, the time span to reach the first minimum, which signs a half completed monolayer, is shorter that required to complete the monolayer. In addition, the slope of decrease following the peak is weaker that observed at start of deposition.

The explanation can be found in the evolution of the sticking coefficient, which can be inferred from the flux of desorbed species. Figure 1b shows the signal collected during deposition by a Quadruple Mass Spectrometer (QMS) facing the sample (see figure S1), this signal is the sum of the intensities measured at m/z= 252 (intact perylene), 125 and 126 (fragments generated in the ionization zone of the QMS). Assuming that the sticking coefficient is initially unity, it starts to decrease as soon as the reflectivity reaches its minimum. Following completion of the wetting layer, the flux of desorbed molecules increases more rapidly. This behaviour points to the existence of repulsive intermolecular forces induced by a charge transfer with the substrate.

The reflectivity curve tells how many particles are scattered within the specular cone, it essentially measures the density of topographic defects. A more refined step consists in analysing the intensity distribution of particles that actually fall within this specular cone. In particular, the mean exit angle is very sensitive to subtle variations in the local molecular arrangement. The time evolution of this parameter is shown in figure 1c. Besides the large increase due to growing roughness as coverage approaches half a monolayer, weaker yet significant variations are visible at different stages of the film growth. Two kinks, at 1260 and 2050 s respectively, translate a change of the local organization, i.e. most probably phase transitions. The sharp rise-and-drop observed on the reflectivity curve is visible here as a rapid oscillation. A sensitivity of 20 to 30 µrad is achieved on the mean scattering angle. In contrast to X-rays or electrons, a quantitative analysis of the GIFAD pattern yields not only the lattice parameter but also the corrugation of the He-surface interaction potential. [START_REF] Khemliche | Grazing Incidence Fast Atom Diffraction: an innovative approach to surface structure analysis[END_REF] In a first order approximation, the latter can be considered proportional to the electron density. [START_REF] Esbjerg | Dependence of the He-scattering potential at surfaces on the surface-electron-density profile[END_REF][START_REF] Nardelli | A density-functional study of van der Waals forces: He interaction with a semiconductor surface[END_REF] Thus the diffraction pattern depends directly on the electronic corrugation as it could be determined by Atomic Force Microscopy (AFM) in contact mode. Due to the grazing geometry, there is an averaging effect along the beam direction and it is therefore the mean corrugation taken over many lattice units that finally produces the observed diffraction pattern.

At the end of the growth process described in figure 1, GIFAD measurements performed by rotating the sample azimuth reveal two distinct crystalline structures, we name them S1 and S2.

The former is characterized by a higher molecular density and is comparable to that reported by Gao and co-workers. [START_REF] Gao | Understanding and controlling the weakly interacting interface in perylene/Ag(110)[END_REF] For the same system, Bobrov et al. [START_REF] Bobrov | True perylene epitaxy on Ag(110) driven by site recognition effect[END_REF] have derived from calculations a similar structure as the most compact crystalline lattice. The detailed description of these structures is well beyond the scope of this report; the most relevant information to retain is that S1 is more compact than S2.

To help identifying the actual nature of the sharp transition visible in both reflectivity and mean exit angle, we performed a new growth sequence on a freshly prepared substrate. This time, the He beam is aligned at 71.5° from the Ag [1-10] direction, i.e. along a mean crystallographic direction of the structure S1. To increase resolution and contrast, both beam energy and incidence angle have been reduced to 300 eV and 0.5° respectively. Figure 2a shows the corresponding reflectivity curve; the main features of figure 1 are well reproduced. After closing the shutter, a clear diffraction pattern starts to appear (see the multimedia file); figure 2b shows the image resulting from summing the 5 last images of the sequence. On this image, one can actually distinguish two types of features; at the lower part of the scattered pattern, two side spots are observed in addition to the 0 th order, this diffraction pattern corresponds to structure S1. Above it, on the right hand side lies another spot that belongs to structure S2 whose crystallographic axis is misaligned by few degrees. For this reason, the associated diffraction pattern becomes asymmetric [START_REF] Zugarramurdi | Surface-grating deflection of fast atom beams[END_REF] with Bragg peaks distributed on a larger Laue circle whose center is shifted horizontally as represented by the dash lines in figure 2c. Another image, showing more clearly the diffraction pattern arising from both structures S1 and S2 and recorded at a slightly larger incidence angle, is shown in figure S4.

We now reanalyse the deposition sequence using the following procedure. We select a region in the ( , ) plane (see angle definition in figure S1) that contains one of the features discussed above; this is sketched in figure 3a. The data set is a 3 dimensional matrix = ( , , ) where t represents the time and I the intensity. For each selected zone, the data is integrated within the corresponding band and the intensity is then reduced to = ′( , ). The result of this treatment is shown in the upper part of figures 3b and 3c. At start, only the 0 th diffraction order from the substrate is visible since the He beam is not aligned with any particular crystallographic direction of Ag(110). In this condition, the electronic density averages to a flat profile. At the reflectivity peak, the 0 th order reappears on top of a diffuse background.

On these graphs, one already notices that diffraction, meaning long range order, appears earlier from S2 than from S1. We conclude that S2 forms before the reflectivity peak, whereas S1 appears following the sharp rise-and-drop of the reflectivity. To better visualize the relative intensity evolution of the diffraction spots, the data is replotted after normalizing the intensity with respect to the 0 th order. The results are shown in the lower part of figure 3b and3c, with the reflectivity curve superimposed. For structure S1 (figure 3b), the striking evidence is the instantaneous appearance of high order diffraction peaks right near the end of the reflectivity drop, just before closing the shutter. By contrast, for structure S2 (figure 3c) high order peaks start to appear well before the reflectivity peak.

On the basis of the experimental elements described in the previous section and complementary information derived from the literature, we propose a model describing the successive steps leading to crystallization of the perylene monolayer into a compact structure. Such a dynamics can be divided in three stages, as depicted in figure 4a. The first stage, which spans up to half way between the reflectivity extrema (the first kink in the mean scattering angle) consists in the building up of a dilute gas-like phase with molecules lying flat on the substrate; 17 molecules are very mobile and segregation does not occur. At the end of this stage, the sticking coefficient starts to decrease (figure 1b) and the kink in the mean exit angle (figure 1c) could be the sign of a transition from the gas to the liquid phase. In the second stage, crystalline domains start to form as high order diffraction peaks appear in structure S2 (figure 3c). At this stage, there is coexistence between liquid and crystalline (under a low density structure) domains, as reported by Bobrov et al. [START_REF] Bobrov | True perylene epitaxy on Ag(110) driven by site recognition effect[END_REF] At the reflectivity peak, this mixed monolayer is characterized by a good flatness as evidenced by the high intensity of reflected particles and the appearance of a specular (0 th order) peak (figure 3b).

In figure 1, concomitant to the reflectivity loss around 2000 s, we observe simultaneously a change of slope in the mean exit angle and a sharp increase in the flux of desorbing molecules. Although an order-disorder transition may accompany the densification of the first layer, as has been reported by Weidong Dou et al., [START_REF] Dou | Aggregation of organic molecules on silver surface with the balance between molecule-substrate interaction and intermolecular interaction[END_REF] this would rather be the signature of a nearly closed monolayer and the start of a second layer. This interpretation is confirmed by the fact the diffraction features from structure S2 do not degrade with decreasing reflectivity (figure 3c).

Note that, at the end of this second stage, the flux of desorbing molecules increases substantially, meaning that repulsive forces prevent landing molecules from adsorbing in the first layer. For this reason, in a third stage, they rather adsorb in a second layer, inducing a loss of reflectivity.

Next, densification of the remaining liquid domains of the first layer proceeds through incorporation of second layer molecules. When a critical density in the first layer has been reached, the associated change of morphology triggers a change of conformation of the molecules in the second layer, these undergo a transition from planar to tilted position. With respect to the incident He beam, the scattering cross section is thus greatly enhanced and the reflectivity drops. In turn, this allows crystallization, possibly through orientational ordering, of the first layer into a compact structure. Note that in bulk crystalline perylene, molecules do not adopt a parallel structure but rather a herringbone type ordering. [START_REF] Fedorov | Structural and electronic properties of perylene from first principles calculations[END_REF] On Ag(110), as the density increases within the first layer, the subsequent change of conformation that may consist in a hexatic phase, modifies the molecules-surface interaction, which in turn triggers a reorientation of molecules of the second layer. This interplay between first and second layer conformations seems to be a general feature of organic layers; it has for instance been reported on bilayers of sexiphenyl molecules on Cu(110), [START_REF] Wagner | Revealing the buried metal-organic interface: restructuring of the first layer by van der Waals forces[END_REF] α-6T on Ag(111) [START_REF] Ghanbari | Layer-resolved evolution of organic thin films monitored by photoelectron emission microscopy and optical reflectance spectroscopy[END_REF] and alkane films on the native oxide layer of a silicon substrate. [START_REF] Corrales | Spontaneous formation of nanopatterns in velocity-dependent dip-coated organic films: from dragonflies to stripes[END_REF] Grazing Incidence fast Atom Diffraction (GIFAD) has been used to follow in real time the organization dynamics of a perylene monolayer on Ag(110). This original technique permits sensitive characterization of the growing layer in terms of the nanoscopic roughness, changes in the local molecular arrangement and crystalline order without perturbing the growth process. The most spectacular results is the observation of a quasi-instantaneous crystallization of the first layer into a compact and very stable structure. We suggest a model through which an intimate interplay between the nearly closed first layer and the growing second layer produces a densification of the former, a change of conformation in the latter and in turn crystallization of the first layer. Although perylene-Ag interaction is large enough to impose some epitaxial relationship in the low density monolayer, [START_REF] Bobrov | True perylene epitaxy on Ag(110) driven by site recognition effect[END_REF] intermolecular Van der Waals forces take over at higher density and direct the selforganization. The bilayer can then be considered decoupled from the substrate.

Remarkably, the mean scattering angle of the probe beam may be treated as a thermodynamic order parameter, it senses small variations in the statistical average of the local molecular arrangement. Thus we are able to clearly identify gas-to-liquid and liquid-to-solid phase transitions; at first sight, the ultra-fast transition observed here has all the properties of a first-order transition. Another impact of this study has to do with the thermodynamics of phase transitions in 2D systems. [START_REF] Zangi | Phase transitions in a quasi-two-dimensional system[END_REF] Contrary to 3D systems where first order structural phase transitions are commonly observed, [START_REF] Ni | First-order structural phase transition in CaFe2As2[END_REF] the lack of long range translational order in 2D system has given rise to a controversy as to whether the liquid-solid transition requires a two-step process implying an intermediate hexatic phase. The latter is characterized by a quasi-long range orientational order and a shortrange translational order. The two-stage model proposed in figure 4 for describing the rise and drop may be compatible with an intermediate hexatic phase.

There exist in fact a large variety of phases, besides the generic gas-liquid-solid triad. Organic layers are good candidate for exploring these different phases; first because they are more likely (like noble gas layers) to build up layers that are largely decoupled from the substrate and second because the molecular orientation and intermolecular interactions provide an extended phase space for their mode of organization. Finally, besides providing a sensitive control over the growth mode and thus the possibility to greatly improve the quality of organic/inorganic interfaces, the delicate dynamics revealed by GIFAD may also represent a guide to improve our description of dispersion forces in these systems.

Supporting Information

• Experimental method; experimental setup: figure S1; reflectivity curve measured with a moderate quality substrate: figure S2; reflectivity curve over an extended deposition time: figure S3; diffraction pattern measured at a larger incidence angle following the growth sequence described in figures 2 and 3 : figure S4 (file Supplementary Information-Momeni.pdf)

• Movie from the growth sequence described in figures 2 and 3 (Growth-sequence-Momeni.avi)

ACKNOWLEDGMENT

We would like to thank Marie Labeye and Huda Alsalem for their participation to part of this work.

We acknowledge financial support from CNRS and University Paris-Sud.

Figure 1 .

 1 Figure 1. Deposition probed by 500 eV He at 1° incidence. a) Reflectivity curve relative to the bare substrate, the sequence contains 1500 images of 3 s. exposure; b) Flux of desorbing molecules obtained by summing the signals measured at m/z=252 (intact molecules), 125 and 126 (fragments generated in the QMS; c) Time evolution of the mean polar exit angle.

Figure 2 .

 2 Figure 2. Deposition probed by 300 eV He at 0.5° incidence. a) Reflectivity curve relative to the bare substrate, the sequence contains 300 images of 15 s exposure. b) Diffraction pattern obtained by summing the 5 last images of the sequence; c) it contains diffraction peaks characteristic of both S1 and S2 structures.

Figure 3 .

 3 Figure 3. Same data as in figure 2 but treated by zones in the scattered intensity. a) zones in the polar angle θ used to reduce the data set. b) and c) time evolution of the azimuthal angle φ for structure S1 and S2 respectively; in the lower plots the intensity has been normalized to that of the 0 th order peak.

Figure 4 .

 4 Figure 4. a) Proposed scenario leading to crystallization of the perylene monolayer. 1: dilute gas phase; 2: coexisting liquid and low density crystalline domains (S2); 3: landing molecules start to form a second layer, with a reduced sticking coefficient. b) substeps in phase 3 leading to crystallization under S1; a): incorporation of 2 nd layer molecules into the 1 st layer increases its density until a critical value that b) lifts up the second layer molecules and further c) triggers an instantaneous crystallization of the first layer.