Morgane Dolez

Jean-François Nicolas

Estelle Hirsinger
email: estelle.hirsinger@pasteur.fr

Laminins, via Heparan Sulfate Proteoglycans, participate in zebrafish myotome morphogenesis by modulating the pattern of Bmp responsiveness

Keywords: pioneers, medial fast fibres, engrailed, phospho-Smad, sleepy, dorsomorphin, Heparinase

In zebrafish, Hedgehog-induced Engrailed expression defines a muscle fibre population that includes both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues such as motoneurons and lateral line. This Engrailed-positive population is restricted in the myotome to a central domain. To understand how this population is established, we have analyzed the phenotype of the sly/lamc1 mutation in the Laminin γ1 chain that was shown to specifically affect Engrailed expression in pioneers. We find that the sly mutation affects Engrailed expression in the entire central domain and that Hedgehog signalling does not mediate this effect. We show that Bmpresponding cells are excluded from the central domain and that this pattern is modulated by Laminins, but not Hedgehog signalling. Knock-down of Bmp signalling rescues Engrailed expression in the sly mutant and ectopically activates Engrailed expression in slow and fast lineages in wild-type embryos.

Lastly, extracellular matrix-associated Heparan Sulfate Proteoglycans are absent in sly and their enzymatic removal mimics the sly phenotype. Our results therefore show that Laminins, via Heparan Sulfate Proteoglycans, are instrumental in patterning Bmp responsiveness and that Bmp signalling restricts Engrailed expression to the central domain. This study underlines the importance of extracellular cues for the precise spatial modulation of cell response to morphogens.

INTRODUCTION

Production of muscle fibre populations with distinct characteristics is essential for the morphogenesis of a functional myotome. In zebrafish, Engrailed expression defines a muscle fibre population that is restricted in the myotome to a central domain. This Engrailed-positive population encompasses both slow and fast fibre types and exhibits an organisational role on myotome and surrounding tissues such as motoneurons and lateral line. The aim of this study is to understand how this Engrailed-positive central domain is established.

A zebrafish primary myotome is composed of two major skeletal muscle fibre types, the superficial slow muscle fibres and deep fast muscle fibres (reviewed in [START_REF] Ochi | Signaling networks that regulate muscle development: lessons from zebrafish[END_REF]. Engrailed transcription factor is expressed in a subset of both fibre types. Engrailed-positive pioneer slow muscle fibres and medial fast fibres are grouped together in a central domain of the myotome [START_REF] Hatta | Diversity of expression of engrailed-like antigens in zebrafish[END_REF][START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. Engrailed expression thus defines a muscle fibre population that encompasses two muscle fibre types. Engrailed-expressing pioneers exhibit an unusual shape, stretching from the somite lateral surface to the notochord. Low-Engrailed-positive medial fast fibres (MFFs) surround the pioneers. While fast and slow muscle fibres constitute the operational musculature of the swimming larva, pioneers are a particular cell population in that their position and morphology prefigure the horizontal myoseptum [START_REF] Felsenfeld | The fub-1 mutation blocks initial myofibril formation in zebrafish muscle pioneer cells[END_REF]. The pioneer presence is associated with the formation of the horizontal myoseptum that gives zebrafish somites their characteristic chevron shape [START_REF] Van Eeden | Mutations affecting somite formation and patterning in the zebrafish, Danio rerio[END_REF] and constitutes a myotendinous junction, functionally analogous to the mammalian tendon [START_REF] Bassett | The zebrafish as a model for muscular dystrophy and congenital myopathy[END_REF].

Hedgehog (Hh) signalling, originating from the notochord, has been shown to commit slow muscle precursors to their slow and pioneer fates and to differentially activate engrailed expression in these precursors [START_REF] Hirsinger | Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors[END_REF][START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. Specification of pioneer precursors requires a higher concentration and longer exposure to Hedgehog signalling compared to slow precursors (reviewed in [START_REF] Ochi | Signaling networks that regulate muscle development: lessons from zebrafish[END_REF]. It is unclear how this is achieved considering that both cell types derive from the same pool of precursors, the adaxial cells which are expected to be all exposed to similar Hedgehog signalling. Indeed, adaxial cells are organised in a transient epithelial monolayer abutting the notochord; it is when they are anchored in the peri-notochordal basement membrane that they commit to their fates [START_REF] Hirsinger | Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors[END_REF]. As the somite matures, they reorganise to stack up on one another while elongating on the antero-posterior axis and then slow muscle fibres migrate across the field of fast muscle precursors to reach their final superficial location [START_REF] Devoto | Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation[END_REF].

Low-Engrailed-positive medial fast fibres are specified in an Hh-dependent manner. They appear in the vicinity of the pioneers, at a later stage when the slow muscle fibres have migrated laterally [START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. The mechanisms that delay engrailed activation and limit it to a subset of fast muscle precursors are not understood.

The sly/lamc1 mutation disrupts the gene for Laminin γ1 chain. Engrailed expression is specifically affected in pioneers at 24 hours post-fertilisation (hpf); basement membranes are disorganised and non-functional and somites are mis-shaped [START_REF] Parsons | Zebrafish mutants identify an essential role for laminins in notochord formation[END_REF][START_REF] Stemple | Mutations affecting development of the notochord in zebrafish[END_REF][START_REF] Wiellette | Combined haploid and insertional mutation screen in the zebrafish[END_REF]. Laminins are extracellular matrix α x β x γ x trimeric proteins, the deposition of which (in particular that of Laminin111) is the first and necessary step to assemble basement membrane (reviewed in [START_REF] Li | The role of laminin in embryonic cell polarization and tissue organization[END_REF]. Laminin γ1 chain is part of 10 Laminins out of 18, including Laminin111. Laminins are found in close proximity to developing muscle cells, i.e. in the perinotochordal basement membrane, around forming somites and then, around myotomes including vertical myosepta [START_REF] Parsons | Zebrafish mutants identify an essential role for laminins in notochord formation[END_REF][START_REF] Snow | Dynamic formation of microenvironments at the myotendinous junction correlates with muscle fiber morphogenesis in zebrafish[END_REF].

To understand how the Engrailed-positive central domain is established, we have studied the sly/lamc1 mutation. Our study shows that it is Bmp signalling that restricts Engrailed expression to the central domain and that this role requires Laminins, via Heparan Sulfate Proteoglycans, to adequately pattern the Bmp responsiveness in the myotome.

MATERIALS AND METHODS

Fish strains

Wild-type AB and sleepy/lamc1 wi390 [START_REF] Wiellette | Combined haploid and insertional mutation screen in the zebrafish[END_REF] embryos were obtained from zebrafish (Danio rerio) lines maintained according to standard procedures [START_REF] Westerfield | The Zebrafish Book: A guide for the Laboratory Use of Zebrafish (Danio rerio)[END_REF]. Embryos were staged by hours post-fertilization (hpf) and by standard staging criteria [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF]. sly embryos are morphologically identifiable from the 18s (somite) stage. For earlier stages and morpholino-injected embryos, the ratio of embryos with a given phenotype was compared to the expected mutant ratio of 1/4.

lamc1 morpholino injections

To target the maternal/residual contribution of lamc1, a translation-blocking morpholino (MO) [START_REF] Nasevicius | Effective targeted gene 'knockdown' in zebrafish[END_REF] was injected into embryos from an incross between sly heterozygote parents. 1/4 of the embryos are therefore depleted from lamc1. The MO (Gene Tools, LLC) used against lamc1 was: 5'-TGTGCCTTTTGCTATTGCGACCTC-3' [START_REF] Parsons | Zebrafish mutants identify an essential role for laminins in notochord formation[END_REF]. A volume of 1,5 nL of a 300 μM MO solution was injected into the cell of one-cell stage embryos.

In situ hybridization and probes

Whole-mount in situ hybridization reactions were carried out as previously described [START_REF] Thisse | Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos[END_REF]. Riboprobes used detected eng1a, eng2a [START_REF] Ekker | Coordinate embryonic expression of three zebrafish engrailed genes[END_REF], eng1b [START_REF] Amores | Zebrafish hox clusters and vertebrate genome evolution[END_REF] and ptc1 [START_REF] Concordet | Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning[END_REF]. Images were acquired using a Leica MZFLIII combined to a Zeiss AxioCam digital camera.

Cyclopamine treatment

Cyclopamine treatment followed a standard method of immersion of chorionated embryos in a 100 μM cyclopamine solution (Toronto Research Chemicals, cat# C988400, dissolved in Embryo Medium and 0,5% ethanol) from the sphere stage [START_REF] Hirsinger | Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors[END_REF]) until fixation at the 18-20s stage.

Dorsomorphin treatment

To avoid biological side effects of DMSO, dorsomorphin (SIGMA, cat# P5499) [START_REF] Yu | Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism[END_REF] was protonated with an excess of HCl, lyophilized and then re-dissolved in fish water. Dechorionated embryos were immersed in a 30 μM dorsomorphin solution in fish water from the 6s stage until fixation at 26 hpf. As expected, a subset of dorsomorphin-treated embryos exhibited ectopic tails, which result from Bmp down-regulation (WT n=26/38, sly n=16/25) [START_REF] Pyati | Transgenic zebrafish reveal stage-specific roles for Bmp signaling in ventral and posterior mesoderm development[END_REF][START_REF] Yu | Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism[END_REF].

Heparinase III and Chondroitinase ABC treatments

Heparinase III (SIGMA, cat#H8891) was dissolved at 0.4-0.5 IU/mL in 20 mM Tris-HCl (pH 7.85), 0.5 mg/ml bovine serum albumin and 4 mM CaCl2 (activator). Chondroitinase ABC (SIGMA, cat#C3667) was dissolved at 5 IU/mL in 50mM Tris-HCl (pH 7.85), 0.25 mg/mL bovine serum albumin and 60 mM Na Acetate (activator). To monitor the injection, the solutions contained 0,5% of phenol red.

Embryos at the 6 or 10s stage were dechorionated and mounted on their lateral side in a drop of lowmelting agarose (1% in Embryo Medium) covered by Embryo Medium on a depression slide. Under a Leica S8Apo stereomicroscope, an approximate total volume of 30-50 nL was injected at three locations (in the anterior and mid-presomitic mesoderm and in the tail bud) using a Marzhauser MM-33 micromanipulator and a MPPI II (Applied Scientific Instrumentation) pressure injector. The embryos were un-embedded, incubated in Embryo Medium and analyzed after varying intervals (5-12 hours post-injection).

Antibodies and immunostaining

Antibodies used were: monoclonal anti-Myosin F59 (1:5 on sections; DSHB), polyclonal anti-Laminin (1:400 on sections and 1:200 in toto; SIGMA, cat# L-9393), polyclonal anti-phospho-Smad1/5/8

(1:100 on sections and 1:50 in toto; Cell Signaling Technology, cat #9511), monoclonal anti-Engrailed 4D9 (1:50 on sections and 1:25 in toto; Santa Cruz Biotechnology, clone sc-53019), polyclonal anti-Prox1 (1:100 in toto; RELIATech GmbH, cat# 102-PA32AG), monoclonal anti-Heparan Sulfate IA 52242. The Laminin antibody recognizes at least Laminin111 in zebrafish [START_REF] Parsons | Zebrafish mutants identify an essential role for laminins in notochord formation[END_REF].

The anti-Heparan Sulfate antibody recognizes the glycosaminoglycan moiety of the Heparan Sulfate Proteoglycans [START_REF] David | Developmental changes in heparan sulfate expression: in situ detection with mAbs[END_REF]; similarly, the anti-Chondroitin Sulfate antibody is immunospecific for an octasaccharide motif present in Chondroitin Sulfate-containing Proteoglycans [START_REF] Ito | Structural characterization of the epitopes of the monoclonal antibodies 473HD, CS-56, and MO-225 specific for chondroitin sulfate D-type using the oligosaccharide library[END_REF].

Secondary antibodies were Alexa-Fluor 488-, 546-and 568-conjugated goat anti-mouse and goat antirabbit antibodies (1:200; Invitrogen).

Immunostaining on cryosections (16μm) was performed as previously described [START_REF] Devoto | Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation[END_REF].

For whole-mount immunostaining with the Laminin antibody, embryos were fixed in 4% formaldehyde overnight at 4°C and then stored in methanol, followed by several washes in PBS-Tween 0,1%, digestion in Proteinase K (10 μg/mL) for 1 minute at tail-bud stage and post-fixation in 4% formaldehyde for 20 minutes at room temperature.

For other antibody staining, embryos were fixed in 4% formaldehyde for 4 hours at 4°C followed by cold acetone treatment during 10 min and then blocking for 30 min in PBDTx (1% BSA, 1% DMSO, 0,5% Triton X-100 in PBS) -2% goat serum. Staining was conducted in PBDTx.

For both staining, primary antibody was incubated overnight at 4°C and secondary antibody for 5 hours at room temperature.

Sections (mounted in DAPI-containing Vectashield, Vector cat# H-1200) and embryos were observed and images were acquired using a Nikon Digital Sight DS-5mc digital camera mounted on a Nikon Eclipse 90i microscope (20X and water-immersion 40X objectives).

For the lateral view on Fig. 3 and detail images on Fig. 5, 6 and S1, embryos were mounted in Vectashield (Vector, cat# H1000) and images acquired with 40X oil-immersion objective under a Leica SP5 confocal microscope.

Quantification and statistical analysis of numbers of pioneer and MFF nuclei

To buffer the natural variability in Engrailed+ nuclei number from one somite to another, nuclei of Prox1-/Engrailed+ MFFs and Prox1+ /Engrailed+ pioneers were counted over x somites in n embryos at two axial levels, trunk (somites 9-11) and tail (somites 18-22) under a Nikon Eclipse 90i microscope. The statistical significance of the difference of nuclei number distributions between two conditions were assessed using the two-tailed Mann-Whitney U test (GraphPad Prism5 software).

RESULTS

Engrailed expression is absent from the entire central domain in the sly mutant

Pioneer and MFF precursors develop in close proximity to Laminin-rich extracellular matrices (Fig. 1A,C). In the sly mutant [START_REF] Parsons | Zebrafish mutants identify an essential role for laminins in notochord formation[END_REF][START_REF] Stemple | Mutations affecting development of the notochord in zebrafish[END_REF][START_REF] Wiellette | Combined haploid and insertional mutation screen in the zebrafish[END_REF], Laminin staining is absent from these sites and Engrailed expression is absent from pioneers (Fig. 1B,D). We therefore assessed the presence of Engrailed in the MFFs, the other component of the central domain.

In 26 hpf wild-type embryos in the anterior somites (n=26/26), Engrailed is expressed at a low level in MFFs (arrows) and at a higher level in pioneers (arrowheads) (Fig. 1E and 5A-D) (this study; [START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. In 26 hpf sly embryos (n=55/55), Engrailed expression at wild-type level is absent (Fig. 1F and 5E-H). In a subset of the embryos (n=28/55), weak Engrailed expression is detected in 2 to 6 cells, often in the trunk and at the trunk-tail transition (data not shown). This probably corresponds to a declining level of an earlier expression of three engrailed genes observed in sly embryos up to the 15s stage (Fig. 2A,B,D,E for eng2a; data not shown for eng1a and eng1b). By lamc1 knock-down in sly embryos, we show that this early expression (Fig. 2C,F for eng2a; data not shown for eng1b) is due to maternal and/or residual lamc1 expression (Fig. 2G-I) that disappears by the 5s stage in the sly mutant.

The sly mutation therefore specifically affects Engrailed expression in both the slow and fast muscle fibre types, i.e. in the entire central domain.

Hedgehog signalling is operational in sly embryos

Hh signalling has been shown to regulate engrailed expression in both slow and fast muscle fibre types (reviewed in [START_REF] Ochi | Signaling networks that regulate muscle development: lessons from zebrafish[END_REF]. Its spatio-temporal window of action on pioneers has been delimited to the anterior presomitic mesoderm and last formed somites [START_REF] Hirsinger | Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors[END_REF] while MFFs require Hh signalling only from the 18s stage [START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. Ptc1 is both a receptor of Hh ligands and a target of the Hh pathway [START_REF] Concordet | Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning[END_REF] and as such was used to assess Hh signalling in wild-type and sly embryos up to 24 hpf. ptc1 is first expressed in adaxial cells and then in MFFs as slow muscle fibres migrate laterally [START_REF] Concordet | Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning[END_REF][START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. ptc1 expression in sly is similar to wild-type from the 5s stage to 24 hpf, in particular at the 15s (n=23/23 embryos of a sly clutch), 20s (n=8/8 sly embryos) stages and 24 hpf (n=16/16 sly embryos), even though in some 24 hpf sly embryos, the staining is more diffuse (n=5/16) (Fig. 2J,K,).

In anti-lamc1 morpholino-injected sly embryos (n=10/39 embryos of a sly clutch), despite a slight reduction in the most anterior somites, ptc1 is normally expressed in the rest of the somites and presomitic mesoderm at the 15s stage (Fig. 2L).

Based on ptc1 expression, Hh signalling is not affected in sly embryos.

Pioneers, MFFs and their precursors are unresponsive to Bmp, in a Hedgehog-independent manner A candidate to mediate the sly phenotype is Bmp. Bmp sources are located dorsally and ventrally to the myotome; receptors and intracellular transducers of the Bmp pathway are dynamically expressed in the somite and in particular in adaxial cells and sclerotome [START_REF] Dick | Smad1 and Smad5 have distinct roles during dorsoventral patterning of the zebrafish embryo[END_REF][START_REF] Mintzer | Lost-afin encodes a type I BMP receptor, Alk8, acting maternally and zygotically in dorsoventral pattern formation[END_REF][START_REF] Muller | Characterization of zebrafish smad1, smad2 and smad5: the amino-terminus of smad1 and smad5 is required for specific function in the embryo[END_REF][START_REF] Nikaido | Restricted expression of the receptor serine/threonine kinase BMPR-IB in zebrafish[END_REF][START_REF] Patterson | BMP regulation of myogenesis in zebrafish[END_REF][START_REF] Rissi | Zebrafish Radar: a new member of the TGF-beta superfamily defines dorsal regions of the neural plate and the embryonic retina[END_REF][START_REF] Schmid | Equivalent genetic roles for bmp7/snailhouse and bmp2b/swirl in dorsoventral pattern formation[END_REF] http://zfin.org, expression pattern database).

To learn whether Bmp signals to muscle precursors, wild-type embryos were immunolabeled for phosphorylated Smad 1/5/8 (pSmad), the active form of the Bmp intracellular transducer (reviewed in [START_REF] Massague | TGF-beta signal transduction[END_REF].

pSmad expression was assessed at the 18-20s stage with respect to Engrailed expression in pioneers.

At the adaxial cell pre-stacking level where slow muscle precursors are committing to their fates, none of the adaxial cells express pSmad (n=22/22) (Fig. 3A,bracket). pSmad is however present in a ventral population, likely the sclerotome, and in a medial dorsal population, likely future fast muscle fibres (data not shown). Just anteriorly, where adaxial cells undergo stacking, adaxial cells that translocated dorsally and ventrally show pSmad labelling while adaxial cells that stayed centrally are still pSmad-negative (n=22/22) (Fig. 3B, bracket). These central cells, by their position and number (2-5 cells per somite), are probable pioneer precursors. The pSmad-positive dorsal and ventral adaxial cells become slow muscle fibres and migrate laterally as they down-regulate pSmad expression (data not shown). At a further anterior level, where pioneers are fully differentiated and express Engrailed, pSmad labelling is mutually exclusive from Engrailed labelling (n=16/16) (Fig. 3F).

Remarkably, the interruption in pSmad labelling matches the region where high levels of notochordderived Hh signalling act on adaxial cells. In view of the antagonistic interactions between Hh and Bmp pathways (reviewed in [START_REF] Guo | Signaling cross-talk between TGF-beta/BMP and other pathways[END_REF][START_REF] Hirsinger | Somite formation and patterning[END_REF], we asked whether Hh signalling was responsible for this interruption. pSmad expression was thus assessed at the 18-20s stage in embryos treated with cyclopamine from the blastula stage. This treatment has previously been shown to result in a total loss of Hh signalling in the myotome [START_REF] Hirsinger | Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors[END_REF][START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF] and we indeed confirmed the absence of Engrailed expression in cyclopamine-treated embryos at 26 hpf (n=17/17; data not shown). However, the interruption in pSmad labelling is similar to wild-type (n=10/10) (Fig. 3E, bracket), despite the expected abnormal morphology of the somite. This aspect of the early pattern of Bmp-responding cells is therefore independent of the Hh signalling. These data show that Bmp signalling in the myotome is tightly regulated in space and time, independently of Hh. It is specifically absent from pioneers, MFFs and their precursors throughout their development.

Response to Bmp signalling is ectopically activated in pioneer and MFF precursors in sly

We next addressed the pattern of Bmp responsiveness in sly where Engrailed is not expressed. Unlike wild-type and cyclopamine-treated embryos, pSmad labelling is detected in the entire adaxial cell population at both levels of pre-stacking (n=5/9) and stacking (n=6/8), in 18-20s stage sly embryos (Fig. 3C,D). Since the strength of the sly phenotype is variable in terms of Engrailed expression levels, the embryos where pSmad expression is not significantly ectopically activated may correspond to mutant embryos with residual levels of Engrailed expression.

At a later stage, in 26 hpf sly embryos, pSmad expression is ectopically activated and maintained in the central domain from which MFFs emerge (n=21/21) (Fig. 4D,E,F). Notably, the lateral expansion and then restriction of pSmad expression in the fast muscle population occurs as in wild-type embryos (n=21/21) (Fig. 4E).

Response to Bmp signalling is therefore ectopically activated in sly pioneer and MFF precursors.

Knock-down of Bmp signalling in sly rescues Engrailed expression in pioneers and MFFs

We tested whether inhibition of Engrailed expression in sly is due to an ectopic response to the Bmp signalling. Treatment with dorsomorphin blocks Smads1/5/8 phosphorylation and therefore specifically knocks-down Bmp signalling [START_REF] Yu | Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism[END_REF]. sly embryos were treated from the 6s stage and Engrailed expression was assessed at 26 hpf when MFFs have developed in the trunk.

Compared to sly embryos (Fig. 5E-H) that do not show any Engrailed labelling, treated sly embryos (Fig. 5I-L) show full rescue of Engrailed expression in both pioneers (arrowheads) and MFFs along the entire axis (n=41/41). Numbers of pioneer and MFF nuclei were counted and statistically analysed (Table 1). Except for a lower number of MFF nuclei in dorsomorphin-treated sly somites (statistically significant at p=0.01), the numbers of trunk and tail pioneer nuclei in treated sly somites are similar to those in untreated wild-type somites. Furthermore, the intensities of the Engrailed labelling and the nuclei shape and cell body positions are similar between normal and rescued cells.

In addition, Engrailed expression is activated at the correct time in pioneers while slightly delayed in MFFs. Wild-type Engrailed expression starts in somites 23-25 for pioneers (n=4) and in somites 14-15 for MFFs (n=4). In treated sly embryos, pioneers start to express Engrailed in somites 23-24 (n=3) and MFFs in somites 12-14 (n=3).

Bmp signalling has been shown to delay myogenesis in zebrafish somites [START_REF] Patterson | BMP regulation of myogenesis in zebrafish[END_REF].

However we find that myosin heavy chain expression is synchronously activated within slow or fast muscle populations irrespective of pSmad normal or ectopic expression. Furthermore, Engrailed expression is not detected in older sly embryos (30 hpf) (data not shown). This shows that absence of Engrailed expression in sly is not linked to delayed myogenesis. It is likely that the indirect activation of Bmp signalling in sly (our study) is mild compared to its direct activation via strong overexpression of Bmp2b in the somites [START_REF] Patterson | BMP regulation of myogenesis in zebrafish[END_REF]. Fine-tuning of the levels of Bmp signalling may indeed be crucial to trigger myogenesis delay since wild-type pSmad-positive cells enter myogenesis on time (our study; [START_REF] Patterson | BMP regulation of myogenesis in zebrafish[END_REF].

The sly phenotype is therefore imputable to ectopic activation of the response to Bmp signalling in pioneer and MFF precursors, leading to Engrailed repression in these cells.

Knock-down of Bmp signalling in wild-type increases the number of Engrailed-positive pioneers and MFFs

To test whether Bmp signalling inhibits Engrailed expression in the wild-type context as well, a similar dorsomorphin treatment was conducted on wild-type embryos and analysed at 26 hpf (Fig.

5M-P).

Numbers of pioneer and MFF nuclei were counted and statistically analysed (Table 1). In the trunk, treated somites (Fig. 5M-P) show additional pioneers and MFFs compared to untreated somites (statistically significant at p=0.01). In the tail however, the number of pioneers are not statistically different between treated and untreated somites. The relative intensities of Engrailed labelling between pioneers and MFFs and the shape of the nuclei are similar between ectopic and normal cells.

Nonetheless, the domain where MFFs are found is expanded dorso-ventrally.

In addition, unlike in pioneers, Engrailed expression is prematurely activated in MFFs. Wild-type Engrailed expression starts in somites 23-25 for pioneers and 14-15 for MFFs (n=4). In dorsomorphintreated wild-types, pioneers start to express Engrailed in somites 24-26; MFFs do so in somites 15-18 (n=5), which corresponds to a 3-somite posterior shift.

These results show that Bmp signalling inhibits Engrailed expression in the wild-type context as well.

The presence of Heparan Sulfate Proteoglycans is affected in sly embryos

No direct interaction between Laminins and Bmp has been described. However, the extracellular matrix-associated Heparan Sulfate Proteoglycans (HSPGs) have been shown to affect BMPs bioavailability (reviewed in [START_REF] Umulis | The extracellular regulation of bone morphogenetic protein signaling[END_REF]. We therefore assessed the presence of HSPGs in wild-type and sly embryos.

At the 18-20s stage, at the level of adaxial cell pre-stacking (data not shown) and stacking (Fig. 6A), in wild-type embryos (n=9/9), HSPGs are found around somitic cells but at a much weaker level around adaxial cells. In sly embryos (n=6/6), HSPGs labelling is very weak (Fig. 6B).

At 26 hpf, at the axial level posterior to the emergence of Engrailed-positive MFFs, in wild-type embryos (n=9/9), HSPGs are still present around somitic cells and now strongly in the basement membrane around the notochord and ventral neural tube as well (Fig. 6C). In sly embryos (n=8/8), HSPGs are absent from these sites (Fig. 6D).

HSPGs are therefore at the right place at the right time to potentially regulate BMPs bioavailability and their presence is disrupted in sly embryos.

Heparinase III treatment, but not Chondroitinase ABC treatment, affects Engrailed and pSmad expression in a manner similar to the sly mutant

To test whether altered presence of HSPGs mediate the effect of sly on Bmp signalling, the effect of HSPGs removal from wild-type embryos was assessed on Engrailed and pSmad expression.

Heparinase III cleaves HS chains, thereby eliminating their activity. Vehicle solution-injected (control) embryos showed normal labelling of HSPGs (n=6/6) (Fig. S2A), Engrailed (n=8/8) (Fig. 6E-H) and pSmad (n=8/8) (Fig. 6M,O). HSPGs are efficiently removed 10 hours after Heparinase III injection (n=13/13) (Fig. S2A-D).

After Heparinase III injection at the 10s stage, Engrailed expression was assessed at 26 hpf (i.e. 12 hours post-injection). The majority of the embryos show a total absence of Engrailed expression at both trunk and tail levels (n=24/35) (Fig. 6I-L) while a minority show weaker Engrailed expression or fewer Engrailed-positive nuclei (n=11/35) (data not shown). The intrinsic variability between injections (volume and location) is likely to account for the different effects that we observe. pSmad expression was then assessed at the 20s stage at the adaxial cell pre-stacking and stacking levels after injection at 6s stage (i.e. 5 hours post-injection). pSmad is ectopically activated in the central domain in a subset of the injected embryos (n=4/10) (Fig. 6N). The lower penetrance of the effect on pSmad compared to that of the Engrailed could be due to the shorter lapse of time between injection and analysis.

At 26 hpf after an injection at the 10s stage (i.e. 12 hours post-injection), pSmad is ectopically expressed in the central domain, where MFFs will emerge, in all injected embryos (n=12/12) (Fig. 6P).

Heparinase III treatment therefore leads to the ectopic activation of pSmad in pioneer and MFF precursors, thereby inhibiting Engrailed expression in these cells.

Chondroitin Sulfate Proteoglycans (CSPGs) are also found in sites compatible with a role in restricting BMPs bioavailability (Fig. S1A,C) and at 26 hpf, CSPGs labelling is weak in sly (Fig. S1B,D).

However, Chondroitinase ABC treatment, that efficiently cleaves Chondroitin Sulfate chains, does not affect Engrailed expression (Fig. S1E-L), despite CSPGs removal 10 hours after injection (Fig. S2E-H). CSPGs are therefore not involved in the regulation of Engrailed expression. The late decrease in CSPGs labelling in sly could be a consequence of extracellular matrix disorganisation or altered notochord maturation.

Considering that Heparinase III treatment mimics the sly phenotype and that HSPGs presence is affected in sly, our results are consistent with a role of HSPGs in mediating the effect of sly on Bmp.

DISCUSSION

This study shows that basement membrane Laminins, via Heparan Sulfate Proteoglycans, are instrumental in the spatial modulation of Bmp signalling. This spatial modulation is required for the patterning activity of Bmp signalling on the myotome, which in combination with Hh signalling, leads to the definition of an Engrailed-positive central domain (see model in Fig. 7A).

Laminins, via Heparan Sulfate Proteoglycans, modulate a subset of the pattern of Bmp responsiveness, leading to the definition of the Engrailed-positive central domain

Basement membrane disorganisation, such as the one described in sly [START_REF] Parsons | Zebrafish mutants identify an essential role for laminins in notochord formation[END_REF], has been shown to result in the disruption of Proteoglycan localisation (reviewed in [START_REF] Li | The role of laminin in embryonic cell polarization and tissue organization[END_REF]. This is what we observe in sly embryos when HSPGs and Laminins colocalize in the extracellular matrices.

HSPGs, unlike Laminins, are also found within the somite and somitic HSPG labelling is weak in sly.

Laminins surrounding the somite may thus play an indirect role on HSPG somitic localisation and/or synthesis.

An increasing body of evidence, including in zebrafish [START_REF] Bink | Heparan sulfate 6-o-sulfotransferase is essential for muscle development in zebrafish[END_REF][START_REF] Machingo | A {beta}1,4-galactosyltransferase is required for Bmp2-dependent patterning of the dorsoventral axis during zebrafish embryogenesis[END_REF][START_REF] Mizumoto | Chondroitin 4-O-sulfotransferase-1 is required for somitic muscle development and motor axon guidance in zebrafish[END_REF], show that the extracellular matrix-associated HSPGs have a positive or negative effect on BMPs presentation to their receptors or their range of diffusion (reviewed in [START_REF] Umulis | The extracellular regulation of bone morphogenetic protein signaling[END_REF]. In particular, HSPGs can restrict Bmp diffusion. We show that this occurs in the sly mutant. An additional mechanism whereby Bmp antagonists emanating from the notochord [START_REF] Dal-Pra | Noggin1 and Follistatin-like2 function redundantly to Chordin to antagonize BMP activity[END_REF][START_REF] Furthauer | Three different noggin genes antagonize the activity of bone morphogenetic proteins in the zebrafish embryo[END_REF][START_REF] Kinna | Knockdown of zebrafish crim1 results in a bent tail phenotype with defects in somite and vascular development[END_REF] modulate Bmp signalling may also play a role in this process.

Only the central domain of the pattern of Bmp responsiveness is modulated by Laminins. The lateral expansion of pSmad expression could be associated with the lateral migration of slow muscle fibres. Indeed these two events are spatio-temporally correlated and slow muscle migration has been shown to create a morphogenetic signal that patterns fast muscle fibre elongation in its wake [START_REF] Henry | Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis[END_REF].

Wild-type pSmad expression is restricted to dorsal and ventral domains. Wild-type ptc1 expression is restricted to the central domain [START_REF] Concordet | Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning[END_REF][START_REF] Wolff | Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo[END_REF]. As a consequence, two domains are set-up in the medial somite (see model in Fig. 7B-E This study shows that as in amniotes (reviewed in [START_REF] Hirsinger | Somite formation and patterning[END_REF], the combined and antagonistic patterning activities of Hh and Bmp generate distinct domains in zebrafish: a central domain, separating dorsal (epaxial) and ventral (hypaxial) domains. Despite a different location within the somite, engrailed expression is nonetheless regulated by a similar regulatory network in amniotes [START_REF] Cheng | The epaxialhypaxial subdivision of the avian somite[END_REF] and zebrafish (this study), as it is induced by Hh and inhibited by Bmps. In cell aggregation assays, chick engrailed-1-expressing cells sort out from hypaxial cells, suggesting that the epaxial-hypaxial boundary may represent a true compartment boundary [START_REF] Cheng | The epaxialhypaxial subdivision of the avian somite[END_REF]. Once again, this report underlines that regulatory networks involving Engrailed, BMPs and Hh in various combinations have been conserved to specify compartments (amniote limb bud, drosophila wing imaginal disk).

Bmp signalling blocks cells from entering the Engrailed-positive population

The involvement of Bmp in Engrailed expression regulation is supported by the results of previous work. Knock-down of the TGF-β family member, Radar, expands eng1a expression in somites [START_REF] Kawakami | The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling[END_REF]. Ectopic expression of chick Dorsalin-1 in the notochord inhibits Engrailed expression [START_REF] Du | Positive and negative regulation of muscle cell identity by members of the hedgehog and TGF-beta gene families[END_REF].

In either rescued sly pioneers and MFFs or ectopic pioneers and MFFs, the relative intensities of Engrailed expression between pioneers and MFFs, their nuclei shapes and cell body positions are similar to wild-type cells. This suggests that Bmp signalling operates as a negative switch for the Engrailed-positive muscle fibre population. When the blockade is relieved, cells proceed and develop their characteristic morphological traits like wild-type cells. Cues other than Bmp have to instruct pioneers and MFFs to express different levels of Engrailed and exhibit distinct morphological traits.

These cues may be linked to the slow or fast identity of pioneers and MFFs.

Hh has been signalling to muscle precursors for a prolonged period before it activates Engrailed expression in pioneers and MFFs [START_REF] Concordet | Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning[END_REF]. Our results suggest that Bmp signalling participates in the regulation of the onset of Engrailed expression in MFFs but not in pioneers. When Bmp signalling is knocked-down, ectopic MFFs appear in the dorsal and ventral myotome domains which in wild-type exhibit a lateral expansion of pSmad expression. Bmp signalling knock-down may affect this lateral expansion, allowing ectopic MFFs to activate Engrailed expression at an earlier stage in this domain. However, even if shifted posteriorly in knock-down experiments, Engrailed expression is not activated in MFFs at the same time as in pioneers. Fast muscle myogenesis is delayed compared to slow muscle myogenesis (reviewed in [START_REF] Ochi | Signaling networks that regulate muscle development: lessons from zebrafish[END_REF]. In both muscle fibre types, Engrailed expression is initiated after the appearance of muscle markers such as myosin heavy chains.

Both pioneers and MFFs may thus need to reach a certain level of myogenic differentiation to activate Engrailed expression.

The roles of Laminins in muscle morphogenesis and differentiation

As previously shown [START_REF] Peterson | Hedgehog signaling and laminin play unique and synergistic roles in muscle development[END_REF], we find that myosin heavy chain expression in the fast muscle population is shifted anteriorly by an average of 4-5 somites, while it is initiated around the correct time in slow muscle fibres. We also find that slow muscle fibre migration is delayed in sly (MD, JFN and EH, manuscript in preparation). Furthermore, delayed fast muscle fibre elongation in sly is linked to slow muscle fibre presence [START_REF] Peterson | Hedgehog signaling and laminin play unique and synergistic roles in muscle development[END_REF]. Considering the morphogenetic role of slow muscle migration on fast muscle fibres [START_REF] Henry | Zebrafish slow muscle cell migration induces a wave of fast muscle morphogenesis[END_REF], this delayed migration is likely to account for the global maturation delay of the fast muscle population in sly. Laminins would therefore play an indirect role on myogenesis through regulation of muscle cell behaviour. This is the case in mouse [START_REF] Bajanca | Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo[END_REF]. Laminins are important for maintenance of immature muscle precursors in the dermomyotome, for proper translocation from the dermomyotome into the myotome and then for proper elongation of the differentiating fibres in the myotome.

We show that untreated and dorsomorphin-treated sly somites are short and straight while Heparinase III-injected somites adopt a chevron shape, similar to wild-type somites. This differential phenotype suggests that Laminins, independently from Bmp signalling, play a role in shaping the somite. This could be through two mechanisms. In sly, notochord cells do not vacuolate and therefore do not stretch the embryo along the antero-posterior axis [START_REF] Stemple | Mutations affecting development of the notochord in zebrafish[END_REF]. The somite shortness may mechanically prevent them from adopting the chevron shape. Alternatively, pioneers, anchored in all four Laminin-rich somite boundaries, are associated with the somite chevron shape [START_REF] Ochi | Signaling networks that regulate muscle development: lessons from zebrafish[END_REF][START_REF] Van Eeden | Mutations affecting somite formation and patterning in the zebrafish, Danio rerio[END_REF]. In sly, pioneers may not properly anchor at these sites and somites would thus not adopt a chevron shape.

The potential role in cell connectivity of the Engrailed-expressing central domain

What remains to be understood is the role of Engrailed on the myotome and surrounding tissues. The results of the Heparinase III treatment suggest that Engrailed, even though expressed in the pioneers, is not required for the chevron shape observed in the trunk somites. However considering the injection stage and location, trunk somites may experience an early and transient Engrailed expression that would be sufficient to trigger the Laminin-dependent morphogenetic program leading to proper shaping of the somite.

Muscle fibres anchor into the horizontal myoseptum to use it as a myotendinous junction [START_REF] Bassett | The zebrafish as a model for muscular dystrophy and congenital myopathy[END_REF]. Pioneers and the horizontal myoseptum are also a source of migrating cues for motoneuron axons [START_REF] Melançon | Pathfinding by identified zebrafish motoneurons in the absence of muscle pioneers[END_REF] and the lateral line primordium, future adult sensory organ [START_REF] David | Molecular basis of cell migration in the fish lateral line: role of the chemokine receptor CXCR4 and of its ligand, SDF1[END_REF]. As of today, it is unclear whether this organising role is mediated by Engrailed per se and/or by pioneers.

The ancestral function of Engrailed is probably regulation of connectivity, as in axon pathfinding and targeting and synaptic specificity, through regulation of cell adhesion molecules (reviewed in [START_REF] Gibert | The evolution of engrailed genes after duplication and speciation events[END_REF]. If Engrailed is directly involved, its organising role in the morphogenesis of both the myotome and surrounding tissues would be in line with its ancestral role in cell connectivity.

Along with recent studies, this work reinforces that free diffusion of morphogens triggering gene regulatory networks are not sufficient to account for the morphogenetic processes occurring in the embryo. The landscape of the extracellular matrix locally modulates the cell response to morphogens.

FIGURES LEGENDS

( 1 :

 1 200 on sections; US Biological, 10E4 epitope, cat# H1890), monoclonal anti-Chondroitin Sulfate (1:200 on sections; SIGMA, clone CS-56, cat#C8035). The monoclonal antibody F59 developed by F.E Stockdale was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City,

  pSmad expression was then assessed with respect to Engrailed expression in MFFs at 26 hpf. At that stage, Engrailed expression appears in MFFs in the trunk. MFFs occupy the entire medio-lateral extent of the central domain. Caudally, the pSmad-positive domain is restricted to a medial one cell-thick line interrupted at the level of the notochord (n=24/24) (Fig.4A, horizontal lines). Before the onset of Engrailed expression in MFFs, while slow muscle fibres migrate, the pSmad-positive domain transiently expands laterally in the fast muscle population, but a pSmad-negative central domain is maintained (n=24/24) (Fig.4B, horizontal lines). After completion of slow muscle fibre migration, Engrailed-positive MFFs (Fig.4C, arrow) develop in this central domain that stays devoid of pSmad labelling (n=21/21) (Fig.4C, horizontal lines). Notably, pSmad expression is restricted again to a medial one-cell thick line of fast muscle fibres.

  ): one central domain corresponding to low Bmp / high Hh signalling flanked on either side by a ventral and a dorsal domain corresponding to high Bmp / low Hh signalling. Initially all adaxial cells are located in the central domain. The stacking of adaxial cells moves slow muscle precursors from the central domain to the ventral and dorsal domains. This ends their exposure to Hh which prevents them from becoming pioneers. The central adaxial cells that stay in the central domain are exposed for a longer time to Hh and become pioneers. The combination of adaxial cell stacking and the pattern of Bmp responsiveness thus provides a mechanism to expose pioneer precursors to Hh signalling for a longer time than slow muscle precursors. At a later stage, this medial central domain transiently expands laterally just before MFFs emerge from it. The patterning of Bmp responsiveness again defines a central domain with low Bmp / high Hh signalling and flanking domains with high Bmp / low Hh signalling. Engrailed expression is thus restricted to the central domain of the fast muscle population, allowing the MFFs to form within this domain.

Fig. 1 .

 1 Fig. 1. Pioneer and MFF precursors develop in close proximity to Laminin-rich extracellular matrices and Engrailed expression is absent from pioneers and MFF in sly. (A,B) Transverse sections of 20s stage WT (A) and sly embryos (B) at the level of early adaxial cell stacking. Dorsal is up. F59 (in green) labels adaxial cells and Laminin is in red; nuclei are counterstained with DAPI (in blue). (C-F) Transverse sections of 26 hpf WT (C,E) and sly (D,F) embryos at the level of the anterior tail (C,D) and the anterior trunk (E,F). (C,D) Laminin is in red. Due to the somite chevron shape, vertical myosepta of the neighbour somite (Blue arrows) are visible on these transverse sections. (C-F) 4D9 (in green) labels pioneers (arrowheads, C,E) and MFFs (arrows, E) in WT embryos and is absent in sly embryos (D,F). n: notochord, nt: neural tube. Scale bar: 50 μm.

Fig. 2 .

 2 Fig. 2. eng2a expression in pioneers requires maternal and/or residual lamc1 while ptc1 expression in the caudal part of the embryo is not affected in lamc1-depleted embryos. (A-F) Lateral views of eng2a expression at the 15 (A-C) and 20s stage (D-F) in WT (A,D), sly (B,E) and morpholino-injected sly embryos (C,F). The fraction of embryos displaying the corresponding phenotype is shown on each panel. In 20s stage sly embryos, no (n=3/11) to weak and transient expression (n=8/11) is found in only 2 to 3 caudal somites (Red arrow in E). (G-I) Dorsal views of Laminin labelling (in red, blue arrows) at the tail bud (Tb) stage in WT (G), sly (H), and lamc1 MOinjected sly embryos (I). Dorsal (J-L) and lateral views (M-O) of ptc1 expression at the 15s stage (J-L) and 24 hpf (M-O) in WT (J,M), sly (K,N,O) and lamc1 MO-injected sly embryos (L). Scale bar: 100μm.

Fig. 7 .

 7 Fig. 7. Laminins, via Heparan Sulfate Proteoglycans, participate in myotome morphogenesis by modulating the pattern of Bmp responsiveness. (A) Laminins would pattern Bmp responsiveness via modulation of HSPG localisation. Combined Bmp and Hh signalling would then define an Engrailed-positive central domain. (B-E) Two domains can be distinguished in the medial somite. A central domain would receive low Bmp, high Hh signalling. Ventral and dorsal flanking domains would receive high Bmp, low Hh signalling. Both Engrailed-positive pioneers and MFFs develop within the central domain at successive developmental stages.

Table 1 . Quantification of pioneer and MFF nuclei in the dorsomorphin treatment experiments.

 1 Values represent the range of numbers of nuclei per somite x is the number of somites counted n is the number of embryos counted Asterisks highlight values statistically different (p=0.01) from the untreated WT value

		Untreated WT Treated sly	Treated WT
	Trunk pioneers 2-5	2-4	3-7 *
		x=15 ; n=5	x=15 ; n=5	x=15 ; n=5
	Trunk MFFs	9-15	2-12 *	17-27 *
		x=15 ; n=5	x=14 ; n=5	x=15 ; n=5
	Tail pioneers	2-6	2-5	3-6
		x=24 ; n=5	x=13 ; n=3	x=24 ; n=5

Acknowledgements

We would like to thank Margaret Buckingham for critical reading of the manuscript. We are grateful to Pascal Dardenne for excellent animal care and to the members of the laboratory for stimulating discussions. We are indebted to Christine Chevalier and Olivier Gorgette (Institut Pasteur) for the statistical analysis. We wish to acknowledge the Plateforme d'Imagerie Dynamique, Institut Pasteur, for their excellent imaging service. We are most grateful to Yves Janin (Institut Pasteur) for preparing the protonated form of dorsomorphin. We would like to thank Monte Westerfield for the generous gift of the sly mutant line. Kate Lewis and Monte Westerfield kindly provided the ptc1, eng1a, eng1b, eng2a probes. This research was supported by core funding from the Institut Pasteur and Centre National de la Recherche Scientifique (CNRS) and by the AFM specific grants (n° 12838 and 14162). M. D. was funded by a fellowship from the French Ministry of Research and Education then by an AFM fellowship (n° 14220). J.F.N. is from the Institut National de la Recherche Médicale (INSERM).

(A-E) Transverse sections of 18-20s stage WT (A,B), sly (C,D) and cyclopamine-treated embryos (E) at the level of adaxial cell pre-stacking (A,C,E) and stacking (B,D). Dorsal is up. F59 (in red) labels adaxial cells and nuclear pSmad labelling is in green; nuclei are counterstained with DAPI (in blue).

(F) Lateral view (projection of 11 confocal sections, 0,6 μm z-step) of a 18-20s stage WT embryo, at the trunk level. Anterior is on the left, dorsal is up. 4D9 (in red) labels pioneers and nuclear pSmad labelling is in green. n: notochord, nt: neural tube. Scale bar: 50 μm.


Aurélie Marion

Marcello Brigante
email: marcello.brigante@uca.fr

Gilles Mailhot

A new source of ammonia and carboxylic acids in cloud water: The first evidence of photochemical process involving an iron-amino acid complex

Keywords: Photochemistry, atmospheric water, oxidative capacity, iron complexes

come

A new source of ammonia and carboxylic acids in cloud

water: The first evidence of photochemical process involving an iron-amino acid complex

Introduction

The atmosphere is a complex medium in which atmospheric particles (liquid and solid) are in continuous interaction with the gas phase. In addition to their direct effect on human health and environmental modifications, atmospheric particles are able to absorb and scatter solar radiation modifying the Earth's radiation budget [START_REF] Papadimas | The direct effect of aerosols on solar radiation over the broader Mediterranean basin[END_REF][START_REF] Singh | Absorbing and scattering aerosols over the source region of biomass burning emissions: Implications in the assessment of optical and radiative properties[END_REF][START_REF] Zhang | Aerosol radiative effect in UV, VIS, NIR, and SW spectra under haze and high-humidity urban conditions[END_REF]. Between the compartments of the atmosphere, cloud water droplets have attracted the attention of numerous researchers working in the field of physical-chemistry. In fact, cloud droplets can be considered as a multiphase reactor in which inorganic and organic constituents interact through numerous chemical and photochemical processes [START_REF] Charbouillot | Mechanism of carboxylic acid photooxidation in atmospheric aqueous phase: Formation, fate and reactivity[END_REF]. During the last decade, it has been clearly demonstrated that reactivity of cloud aqueous phase can impact the oxidative budget of the atmosphere. Between the oxidative species present in cloud water, hydroxyl radical (HO • ) is considered as the most important radical due to the ability to oxidize numerous organic constituents [START_REF] Gligorovski | Environmental implications of hydroxyl radicals (•OH)[END_REF][START_REF] Herrmann | Kinetics of aqueous phase reactions relevant for atmospheric chemistry[END_REF][START_REF] Herrmann | Tropospheric aqueous-phase free-radical chemistry: Radical sources, spectra, reaction kinetics and prediction tools[END_REF]. In the past few years, HO • quantification became of great interest to estimate the life-time of organic compounds and numerous spectroscopic and chromatographic approaches have been developed. In the gas phase, the photolysis of ozone can produce electronically excited oxygen atom O( 1 D) which can then react with water to form hydroxyl radicals. Once in the gas phase, HO • can be transferred to aqueous phases (Henry's law constant K H = 3.0 ± 0.2 M atm -1 ) [START_REF] Hanson | Measurement of hydroxyl and hydroperoxy radical uptake coefficients on water and sulfuric acid surfaces[END_REF][START_REF] Mozurkewich | Mechanisms for the release of halogens from sea-salt particles by free radical reactions[END_REF] However, these two possible sources cannot completely explain the concentration of hydroxyl radical in cloud water (also considering the strong reactivity of this radical with organic compounds). In fact, a scarce number of studies measured the production of hydroxyl radicals in cloud water [START_REF] Arakaki | Sources, sinks, and mechanisms of hydroxyl radical (OH) photoproduction and consumption in authentic acidic continental cloud waters from Whiteface Mountain, New York: The role of the Fe(r) (r = II, III) photochemical cycle[END_REF][START_REF] Faust | Aqueous-phase photochemical formation of hydroxyl radical in authentic cloudwaters and fogwaters[END_REF] and information on the oxidizing capacity of cloud water in terms of quantification of HO • and chemical sources is still a subject of debate. Between the different cloud water constituents, hydrogen peroxide (H 2 O 2 ), through its photolysis, is considered as one of the most important sources of HO • [START_REF] Herrmann | Tropospheric aqueous-phase free-radical chemistry: Radical sources, spectra, reaction kinetics and prediction tools[END_REF][START_REF] Yu | Hydrogen peroxide photolysis in acidic aqueous solutions containing chloride ions. II. Quantum yield of HO (Aq) radicals[END_REF]. Iron reactivity also contributes to the production of HO • in atmospheric aqueous phases upon Fe(III) photolysis and Fenton reaction [START_REF] Deguillaume | Transition metals in atmospheric liquid phases: Sources, reactivity, and sensitive parameters[END_REF][START_REF] Faust | Photolysis of Fe (III)-hydroxy complexes as sources of OH radicals in clouds, fog and rain[END_REF][START_REF] Millero | Effect of ionic interactions on the oxidation of Fe(II) with H 2 O 2 in aqueous solutions[END_REF]. However, organic constituents of cloud water such as short-chain carboxylic acids and siderophores are able to generate stable complexes with iron modifying the photoreactivity and as consequence the hydroxyl radical formation efficiency [START_REF] Faust | Photochemistry of aqueous iron(III)-polycarboxylate complexes: roles in the chemistry of atmospheric and surface waters[END_REF][START_REF] Long | Evaluation of modeled cloud chemistry mechanism against laboratory irradiation experiments: The HxOy/iron/carboxylic acid chemical system[END_REF][START_REF] Passananti | Siderophores in cloud waters and potential impact on atmospheric chemistry: Photoreactivity of iron complexes under sun-simulated conditions[END_REF][START_REF] Weller | Modeling the impact of iron-carboxylate photochemistry on radical budget and carboxylate degradation in cloud droplets and particles[END_REF]. In fact, the contribution of iron species (as aquacomplexes or organic complexes) to the formation of hydroxyl radical is still a subject of debate [START_REF] Bianco | A better understanding of hydroxyl radical photochemical sources in cloud waters collected at the puy de Dôme station -experimental versus modelled formation rates[END_REF].

Recently, the presence of amino acids ranging from nM to few µM concentrations has been reported in cloud water collected at the top of puy de Dôme (France) [START_REF] Bianco | Improving the characterization of dissolved organic carbon in cloud water: Amino acids and their impact on the oxidant capacity[END_REF].

Their contribution to the dissolved organic matter has been estimated to be up to ~ 9% and, considering the high stability constant with ferric ions reported in literature, the presence of stable iron-complexes could be expected [START_REF] Perrin | The stability of iron complexes. Part III. A comparison of 1 : 1 ferric and ferrous amino-acid complexes[END_REF].

In the first part of this paper, we investigate the photoreactivity of Fe(III)-aspartic acid complex as probe for iron-amino acid complexes. Aspartate diaion (Asp) was taken into account due to the presence in cloud water and the strong stability constant of complex with ferric ion (log K = 11.4) [START_REF] Perrin | The stability of complexes of ferric ion and amino-acids[END_REF]. The photostability of Fe-Asp complex, considering stability constants reported in literature, is investigated using UV-vis spectroscopy under monochromatic and polychromatic irradiation. Quantum yields of ferrous ions and hydroxyl radical formation rates are determined in the presence or not of hydrogen peroxide to assess the possible impact of iron-aspartate complex photolysis on the oxidative budget of cloud water. Moreover a phototransformation mechanism leading to the formation of short-chain carboxylic acid and ammonia under polychromatic irradiation is proposed.

Material and methods

Chemicals

All chemicals were used without additional purification: Fe(III)-perchlorate (99.9 %), aspartic acid (99.0 %), malonic acid (99 %), ascorbic acid (99.8 %), coumarin (99 %), 7hydroxycoumarin (99 %), sodium hydroxide (99.9 %), perchloric acid (70 %), and hydrogen peroxide were purchased from Sigma Aldrich. Ferrous perchlorate (99 %), ferrozine (97 %), sodium formate (99 %) were purchased from Fluka. Ammonium oxalate (99 %) was purchased from Prolabo. All solutions were prepared in water purified by a Millipore milli-Q device (Millipore αQ, resistivity 18 MΩ cm, DOC < 0.1 mg L -1 ).

Complex preparation and spectroscopic characterisation

Fresh solutions of Fe-Asp complex are prepared before each experiment by mixing appropriate volumes of Fe(ClO 4 ) 3 and aspartate stock solutions (each 10 mM). The natural pH of mixture was 3.7 and was adjusted using sodium hydroxide if necessary.

In Figure 1 UV-vis spectrum of Fe 3+ , Asp and Fe-Asp (100 µM + 200 µM) solutions in milli-Q water at pH 3.7 are presented showing the overlap with lamp emission spectra (see irradiation experiments description). Complex speciation is predicted using Hyperquad Simulation and Speciation (HYSS2009) software [START_REF] Alderighi | Hyperquad simulation and speciation (HySS): a utility program for the investigation of equilibria involving soluble and partially soluble species[END_REF]. Hydrogen peroxide was added to the solution just before each experiment in order to avoid a dark reactivity in the presence of iron (i.e. Fenton reaction).

Irradiation Experiments

Photochemical experiments are performed under monochromatic and polychromatic irradiation to investigate the Fe-complex disappearance and formation of ferrous ions, hydroxyl radicals and transformation products. For monochromatic irradiation, the procedure is the following: 3 mL of solution are irradiated in a quartz cuvette (Hellma QS) under monochromatic light (313 and 365 nm) using a Hg 200W lamp (Oriel) equipped with a monochromator. At fixed interval time the cuvette is transferred into a Varian Carry 300 UVvis spectrophotometer to follow the time evolution of UV-vis spectra. The radiation fluxes reaching the solution at 313 and 365 nm are respectively 516 and 1230 µW cm -2 . Sunsimulated irradiations are performed in homemade photochemical reactor equipped with four fluorescent lamps (Sylvania Blacklight F15W/350BL) with emission from 290 to 450 nm (centered at 350 nm).

To investigate the photochemical behavior under polychromatic radiation, 250 mL of solution are irradiated in a jacked cylindrical reactor (length = 10 cm, volume = 30 mL) thermostated at 283 ± 2 K using a circulation cooling system under aerated conditions. The total irradiance reaching the solution in the UV region (290-400 nm) is equal to 1.27 mW cm -2 that is closed to the value measured in a sunny day in Clermont-Ferrand (France) (1.23 mW cm -2 ) (see Figure 1)

The emission spectra of the irradiation systems (monochromatic and polychromatic) reaching the solution surface are recorded using an optical fiber with a charge coupled device (CCD) spectrophotometer (Ocean Optics USD 2000 + UV-vis) which is calibrated using a DH-2000-CAL Deuterium Tungsten Halogen reference lamp.

Ferrous ions, hydroxyl radical formation yields and transformation products identification

Ferrous ions (Fe 2+ ) formation is determined using ferrozine complexing agent and detection of complex is performed at 562 nm (ε 562nm = 27900 M -1 cm -1 ) [START_REF] Stookey | Ferrozine -a new spectrophotometric reagent for iron[END_REF] using a Cary 300 UV-vis spectrophotometer.

Hydroxyl radical formation is determined using coumarin as trapping molecules that leads to the formation of stable 7-hydroxycoumarin (7OHC) as high fluorescence product (λ ex = 325 nm, λ em = 456 nm).

Coumarin is added to the solution just before each experiment (a concentration of 500 µM was used to trap all generated hydroxyl radical) and formation of 7OHC is monitored by transferring an aliquot of solution to the spectrofluorometer at fixed interval times. For dark reactivity, coumarin is spiked in a quartz cell previously filled with Fe(III)-Asp + + H 2 O 2 solution. 7OHC formation at different times is directly monitored by putting the cell inside the spectrofluorimeter.

7OHC formation yield (Y 7OHC ) is determined following by HPLC coumarin disappearance and formation of 7OHC using Fenton-like reaction (Fe(III) + H 2 O 2 ) as source of hydroxyl radical. The experiment, presented in Figure SM3, is repeated 4 times at pH 3.7 and 283K. 7OHC formation yield is then estimated as the ratio between formation rate of 7OHC and degradation rate of coumarin for each experiment and an average yield of Y 7OHC is determined to be 4.6±0.5 %. Considering the reactivity constant of hydroxyl radical with coumarin and aspartate (2 × 10 9 M -1 s -1 and 7.5 × 10 7 M -1 s -1 respectively) [START_REF] Buxton | Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (OH/O-) in aqueous solution[END_REF][START_REF] Gopakumar | Gamma irradiation of coumarin in aqueous solution[END_REF] we can estimate that under experimental conditions (highest aspartate concentration of 200 µM and 0.5 mM of coumarin) about 98.5% of photogenerated hydroxyl radicals react with our probe.

The quantum yield of ferrous ions formation is defined as the ratio between the number of ferrous ions formed and the number of absorbed photons during the same period of time.

This value gives an estimation of the photochemical process efficiency. In our experiments iron-aspartate complex is the only absorbing specie present in water and the polychromatic quantum yield formation of Fe(II) ( ()

Fe II 
) can be estimated in the overlap range 290-400 ( 1 and  2 ) by (eq 1). mm) and a metasulfonic acid (MSA) solution as eluent with an isocratic elution for cations.

Laser flash photolysis

The laser flash photolysis system has been previously described [START_REF] Brigante | Photochemistry of 1-Nitronaphthalene: A potential source of singlet oxygen and radical species in atmospheric waters[END_REF] and the only difference is that the current experiments used the fourth harmonic (λ exc = 266 nm) for the excitation (the energy is set to 50 mJ/pulse). An appropriate volume of chemical stock solutions (Fe 3+ and aspartate) is mixed just before each experiment to obtain the desired mixtures and concentrations. All experiments are performed at ambient temperature (293 ± 2 K) and in aerated solutions at pH 3.7±0.2.

Phototransformation experiments

To assess the impact of hydrogen peroxide on the Fe(III)-Asp complex phototransformation, The disappearance of Fe(III)-Asp complex is followed by measuring the absorption at 289 nm while concentration of ferrous ions is determined using the ferrozine method previously described while ammonium ions are quantified with ionic chromatography.

different

Results and Discussion

Complex formation and phototransformation yields

Stability constants of complexes between ferric ion and amino acids have been reported in literature [START_REF] Perrin | The stability of complexes of ferric ion and amino-acids[END_REF] suggesting the formation of stable complex with a stoichiometry 1:1.

High stability constant was found for aspartic acids (logK of 11.4). Vukosav and Mlakar [START_REF] Vukosav | Speciation of biochemically important iron complexes with amino acids: Laspartic acid and L-aspartic acid -glycine mixture[END_REF] investigated the ferric and ferrous ions complexation with an excess of aspartic acid using square wave voltammetry. The authors reported the formation of different iron-aspartate complexes as function of solution pH. In our work, stability constants reported in Table SM1 are used to predict the iron-aspartate complex form under our experimental conditions. In Figure SM1 different forms of iron complexes are reported as function of pH considering 100 µM of Fe(III) and aspartate concentration ranging from 50 to 300 µM. Two main complexes between Fe(III) and aspartate are found : Fe(III)-HAsp 2+ and Fe(III)-Asp + . They both have a stoichiometry ratio of 1:1 and are complexes between Fe 3+

and respectively aspartate monoanion (HL -) and aspartate dianion (L 2-). The latter is expected to be formed increasing the pH in which L 2-is the predominant specie (pKa ~3.8) [START_REF] Rakhimova | Iron aspartate complexes and model processes of their formation[END_REF]. In the presence of aspartate concentration greater than Fe(III), the main specie present in solution at pH 3.7 (natural pH) is Fe(III)-Asp + complex. However, at molar ratio 1:1, ferric ion is partially present as aquacomplex (Fe(OH) 2+ and Fe(OH) 2 + ).

Increasing the concentration of aspartate (ratio 1:2), about 92% of iron is present as Fe(III)-Asp + and no more Fe(II)-aquacomplexes are present. Moreover, no significant difference in the iron speciation can be appreciated using higher ratio (1:3).

Laser flash photolysis experiments are performed using Fe(III) mixed with aspartate solutions and amount of ferric ions disappearance is monitored following the absorption decrease at 290 nm. Iron bleaching is monitored upon 266 nm excitation of iron and iron-aspartate solutions Under monochromatic (313 and 365 nm) and polychromatic (290-450) irradiation, the UV-vis spectrum of Fe(III)-Asp + undergoes a fast decrease, and after about 5 min of irradiation at 365 nm, about 15 % of the complex disappeared (see Figure SM2). Measurements of ferrous ions in solution confirm that photolysis of Fe(III)-Asp + complex leads to the quantitative photoreduction of Fe(III) into Fe(II) and consequent oxidation of ligand following a well-known ligand-to-metal charge transfer reaction (LMCT) as reported in reaction (R1). (3.71 ± 0.23) × 10 -6 M min -1 ) are determined indicating that ferrous ions are stable in water despite a possible oxidation into ferric ions observed at pH higher than 4.0 [START_REF] Morgan | The effect of pH on the kinetics of spontaneous Fe(II) oxidation by O 2 in aqueous solution -basic principles and a simple heuristic description[END_REF]. Direct hydroxyl radical formation has been observed during ironaquacomplexes photolysis while photolysis of iron-organic complexes is suggested to lead to the formation of reactive oxygen species (ROS). Reactivity of dissolved oxygen with organic ligand radical (Asp • ) can lead to the formation of superoxide radical anion (O 2 •-) that can react with iron complex or, through disproportionation, leads to the formation of hydrogen peroxide [START_REF] Bielski | Mechanism of the disproportionation of superoxide radicals[END_REF][START_REF] Wang | Photoinduced degradation of 2,4-dichlorophenol in water: influence of various Fe(III) carboxylates[END_REF][START_REF] Zhang | Photochemistry of the iron(III) complex with pyruvic acid in aqueous solutions[END_REF]. Hydrogen peroxide can react with ferrous ions in solution generating hydroxyl radical (Fenton reaction). Formation of hydrogen peroxide has been also demonstrated during photolysis of Fe(III)-oxalate complexes through oxidation of Fe(II) into Fe(III) by HO 2 [START_REF] Zuo | Kinetics of photochemical/chemical cycling of iron coupled with organic substances in cloud and fog droplets[END_REF].

• /O 2 •-
Zhang and coworkers determined that photolysis quantum yield of Fe(III)-pyruvate complex at 355 nm decreases from 1.0 to 0.46 in deoxygenated and aerated solutions respectively [START_REF] Zhang | Photochemistry of the iron(III) complex with pyruvic acid in aqueous solutions[END_REF]. This result demonstrates the key role of molecular oxygen on the Fe(III)-Asp + is (3.79 ± 0.33) ×10 -6 M min -1 while a rate of (4.99 ± 1.43) ×10 -7 M min -1 and

(2.50 ± 0.17) ×10 -5 M min -1 is determined for Fe(OH) 2+ and Fe(III)-(C 2 O 4 ) + respectively.

In Table 1 Fe 2+ formation quantum yields are reported and compared with previously investigated iron complexes adopted as proxies for Fe-complexes in cloud water. Ferrous ions formation yield from Fe(III)-Asp + complex is about 5 times lower than the value measured for Fe(III)-(C 2 O 4 ) + under the same irradiation conditions. By complexing with organic ligands, Fe(III) photolysis is faster than for the aquacomplex form. This leads to higher Fe 2+ formation rates which modify the speciation of iron in the aqueous phase of cloud and therefore the iron cycle in clouds.

Hydroxyl radical formation

In order to assess the impact of Fe(III)-Asp + complex photolysis on the oxidative capacity and chemical composition of cloud water, different experiments by varying the initial concentration of complex and presence of hydrogen peroxide are performed under dark and polychromatic irradiation as summarized in Table 2. Hydroxyl radical formation rate ( f HO R  ) is quantified spiking the solution with 500 µM of coumarin that quantitatively trap all generated hydroxyl radical ( , coumarin HO k  = 6.4×10 9 M -1 s -1 ) [START_REF] Singh | A pulse radiolysis study of coumarin and its derivatives[END_REF] generating a fluorescent intermediate (7-OH-coumarin, 7OHC).

In dark experiments no relevant formation of hydroxyl radical is observed for all experiments excepted for experiment 2 and 4 in which a formation rate of hydroxyl radical ( f HO R  ) of (2.30 ± 0.12) × 10 -8 and (2.98 ± 0.12) × 10 -8 M min -1 is determined. Under polychromatic irradiation (Figure 5), a similar formation rate of hydroxyl radical is observed for experiment 1 and 3 (ferric ion and Fe(III)-Asp + photolysis) with a f HO R  of (1.06 ± 0.11) × 10 -7 and (9.76 ± 0.44) × 10 -8 M min -1 respectively. In the presence of hydrogen peroxide (1 mM), as expected, an increase of HO • formation rate is observed up to (7.18 ± 0.33) × 10 -7 M min -1 for 100 µM of Fe(III)-Asp + complex in the presence of 1 mM of hydrogen peroxide.

Phototransformation mechanism

The mechanisms of Fe(III)-Asp + complex transformation under cloud water conditions is investigated mixing 5 µM of Fe(III) with 200 µM of aspartate and solution was irradiated under sun-simulated conditions. Fe(III) concentration is typical for aqueous phase of clouds and aspartate concentration is estimated thanks to HYSS2009 software to obtain more than 90% of Fe(III)-Asp + complex. Under these conditions, almost 5 µM of Fe(III)-Asp + are present in solution with an excess of aspartate. In absence of hydrogen peroxide, formations of ammonia (NH 4 + ) and ferrous ions are proportional to the disappearance of Fe(III)-Asp + complex (Figure 6A) suggesting that irradiation of one complex molecule leads to the reduction of ferric into ferrous ion and quantitative formation of one NH 4 + through a complex mechanism starting from a first ligand-to metal charge transfer (LMCT) reaction between Fe(III) and Asp.

When the same experiments are performed with addition of 50 µM of H 2 O 2 (Figure 6B), an important increase of NH 4 + is observed leading to a significantly higher concentration than the initial concentration of Fe(III)-Asp + complex (about 3 times higher after 100 min of irradiation). Moreover, formation of short-chain carboxylic acids such as malonic, oxalic and formic acids is observed during irradiation (Figure SM4). In absence of hydrogen peroxide, carboxylic acids are not detected in solution suggesting that their formation can be probably attributed to the oxidation of aldehyde that is generated from deamination of aspartic radical.

Moreover, no relevant formation of NH 4 + or carboxylic acids has been noticed in absence of ferric ions (i.e. only aspartate and hydrogen peroxide).

The addition of 10 µM of ferric ions after 240 min of irradiation shows an important increase of Fe(III)-Asp + complex which is again photo-degraded and leads to an important formation of NH 4 + . The increase of ferrous ions concentration is not proportional to the formation of ammonia which suggests a re-oxidation of ferrous ions into ferric ions (Fenton reaction)

which is complexed again with the excess of aspartate in solution.

To confirm this hypothesis, the same experiment has been performed with an addition of hydrogen peroxide (200 µM) after 240 min of irradiation. When H 2 O 2 is added (Figure 6C), a relevant decrease of Fe 2+ concentration (about 0.8µM), proportional to the complex formation, is observed confirming a re-oxidation of Fe 2+ into Fe 3+ (Fenton reaction) which then is re-complexed by aspartate in solution. The irradiation of this solution highlights an important increase of ammonia concentration despite a quite stable concentration of Fe 2+ and Fe(III)-Asp + complex. In Figure 7, the proposed mechanisms from Fe(III)-Asp + is presented indicating that different steps leading to the formation of ammonia from Aspartate radical anion. A first decarbonylation of Aspartate radical anion leads to the formation of amine radical. This latter can react with molecular oxygen leading to the formation of ammonia and aldehyde (R-CHO). The formation of short-chain carboxylic acids observed only in the presence of hydrogen peroxide can be attributed to the hydroxyl radical mediated oxidation of aldehyde as reported in the literature [START_REF] Charbouillot | Mechanism of carboxylic acid photooxidation in atmospheric aqueous phase: Formation, fate and reactivity[END_REF]. These different photoinduced steps lead to the formation of carbon dioxide CO 2 and therefore represent a sink for organic compounds in aqueous phase. As observed for Fe-oxalate, by complexation processes and photoreaction iron is allowing large organic compounds to be transformed into smaller compounds and CO 2 .

Conclusions

The present work investigate, for the first time of our knowledge, the photochemical reactivity of an Fe(III)-amino acid complex in atmospheric mimic waters. The photolysis of Fe(III)-Asp + complex under sun-simulated conditions leads to the formation of ammonia as main product and short chain carboxylic acid through a more complex degradation mechanism. In the presence of hydrogen peroxide the formation of hydroxyl radical is enhanced leading to the re-oxydation of ferrous into ferric anion during Fenton process and a possible formation of a new complex. The formation of iron-amino acid complexes can represent a degradation pathway for amino acids oxidation as well as new photochemical sources of carboxylic acids and ammonia in cloud aqueous phase. The relevance of this photochemical reactivity needs to be extended to aerosols. In fact, we can expect that the presence of higher amino acids and iron found in atmospheric particles can induce these new heterogeneous processes enhanced by solar irradiation. For these reasons, iron-amino acids photoreactivty can modify the physico-chemical properties of atmospheric particle (i.e. hygroscopicity) but also the composition of solid and gas phases of atmosphere.

  (II) formation rate (M s -1 ) and I a is the absorbed photon flux per unit of surface and unit of time in the wavelength range by iron-complex. The latter was calculated from is the incident photon flux, ε the molar absorption coefficient of iron-aspartate, l the optical path length inside the cell or the reactor and [Fe(III)-Asp] the initial complex concentration.Ion chromatography (IC) analysis is performed using a Dionex DX-320 apparatus with a KOH gradient elution and equipped with an IonPac AG11 (guard column 4 × 50 mm) and an IonPac AS11 (analytical column 5 × 250 mm) for anions and Dionex ICS-1500 equipped with an IonPac CG16 (guard-column 4 × 50 mm) and an Ion-Pac CS16 (analytical column 5 × 250

  producing a transient spectrum reported in Figure 2A. Interestingly, when experiments are performed in the presence of aspartate high decay is observed at ~290 nm (see insert of Figure 2A) indicating a different photoreduction efficiency of Fe(III) in the presence of amino acid. Absorption decay at 290 nm of Fe(III) [100 µM] solution in the presence of different aspartate concentrations (Figure 2B) also indicates a non-linearity of experimental data fit with a plateau value observed near the ration [Asp]/[Fe(III) = 2.0. For these reasons the next experiments are performed with 100 µM Fe(III) and 200 µM Asp which correspond to the formation of almost 100 µM of Fe(III)-Asp + complex.

  disappearance of complex monitored at 290 nm and formation of ferrous ions under polychromatic irradiation are displayed. Similar degradation and formation rates for Fe(III)-Asp + and Fe 2+

Fe

  (II)/Fe(III) catalytic process in agreement with formation of hydroperoxide/superoxide radical and finally hydrogen peroxide. However, quantification of hydrogen peroxide in solution is difficult due to the fast reactivity with photogenerated ferrous ions. Under polychromatic irradiation ferrous ions formation from 100 µM of Fe(III)-Asp + solution is presented in Figure4and compared to the data obtained from 100 µM of Fe(III)-aquacomplex (Fe(OH) 2+ ) and iron-monoxalate complex (Fe(III)-(C 2 O 4 ) + ). The pH of the solutions is the natural pH : respectively 3.8±0.1 for Fe(III)-Asp+ and Fe(OH) 2 + solutions and 2.6±0.1 for Fe(III)-(C 2 O 4 ) + solution. Under 290-450 nm irradiation Fe 2+ formation rate (

Figure Captions 1 )

 1 Figure Captions

  irradiation experiments are performed in the presence or not of H 2 O 2 . 5 µM of ferric ions are mixed to 200 µM of aspartate before irradiation. The amount of aspartate needed was determined using Hyperquad Simulation and Speciation (HYSS2009) software in order to obtain more than 90% of Fe(III)-Asp + complex in solution. Three experiments are performed: with the presence of Fe(III)-Asp complex in the absence or presence of H 2 O 2 . Moreover, to identify the mechanism in the presence of H 2 O 2 , additions of ferric ions or H 2 O 2 are performed after 240 min of irradiation.

Acknowledgements

Authors acknowledge financial support from the Regional Council of Auvergne and from the "Fédération de Recherches en Environnement" through the C E Environnement founded by the gion Auvergne, the French government, FEDE from the European community, and the ANR BIOCAP (ANR-13-BS06-0004).

Figure 2 23 Figure 6 27