Johnny Beaugrand
email: johnny.beaugrand@inrae.fr

Camille Goudenhooft

Camille Alvarado

Marie-Françoise F Devaux

Camille Rivard

Sylvie Durand

Hugo Chauvet

Matthieu Réfrégiers

Frédéric Jamme

Fabienne Guillon

Fréderic Jamme

Christophe Baley

Alain Bourmaud

Evolution of the flax cell wall composition during development and after gravitropism by synchrotron fluorescence imaging

Keywords: Flax, Infrared microspectroscopy, Hydroxycinnamate, Proteins, Lignin, Polysaccharide

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

Introduction

One of the oldest fibre plants used by humans is a flax plant (Linum Uusitatissimum L.); traces of flax as far as back the prehistoric civilization in Egypt, ancient Babylonia, and Europe [START_REF] Heer | Prehistoric Culture of Flax[END_REF] have been verified. Flax, as industrial crop of economic interest, has been used for textile applications, for new developments in the field of high-performance composites in terms of the mechanics [START_REF] Bourmaud | Towards the design of highperformance plant fibre composites[END_REF][START_REF] Mohanty | Composites from renewable and sustainable resources: Challenges and innovations[END_REF], and for additional functionalities such as acoustic properties, e.g., for violin design [START_REF] Viala | Identification of the anisotropic elastic and damping properties of complex shape composite parts using an inverse method based on finite element model updating and 3D velocity fields measurements (FEMU-3DVF): Application to bio-based composite violin soundboards[END_REF].

Flax belongs to the dicotyledonous group (Ray F. [START_REF] Evert | Esau's Plant Anatomy: Meristems, Cells, and Tissues of the Plant Body: Their Structure, Function, and Development[END_REF], and its development has been well documented and recently reviewed (Goudenhooft et al., 2019a). Basically, this temperate crop follows four identifiable growth stages and can be schemed on a cumulative growing degree-day scale, starting from 0°C as the sowing date and reaching 1,000°C at flax stem fibre maturity. From a description point of view, the beginning is the emergence of the plant, followed by the vegetative stage (VS). At 15-20 days of the VS, the flax plant reaches approximately 15-20 cm (approximately 250 growing degree days).

Then, for approximately three weeks, FG takes place. At that time, the flax plant elongates up to several centimetres per day [START_REF] Gorshkova | Cell-Wall Polysaccharides of Developing Flax Plants[END_REF][START_REF] Heller | A comparative study between Europe and China in crop management of two types of flax: linseed and fibre flax[END_REF], reaching 80-90 cm over this fast FG period (approximately 500 growing degree days).

The flax stem reaches a final length of approximately 1 m [START_REF] Gorshkova | The snap point: a transition point in Linum usitatissimum bast fiber development[END_REF] at fibre maturity (M) (approximately 1000 growing degree days). Some external stresses may impact the bast fibre yield and benefits, such as the lodging phenomenon (Goudenhooft et al., 2019b), a stress of economic importance due to its high impact on farmer incomes. From a fundamental view, it is fascinating to note that flax stems can be restored from lodging through a gravitropism reaction under specific conditions, such as the plant response to gravity after tilting [START_REF] Ibragimova | Development of gravitropic response: unusual behavior of flax phloem G-fibers[END_REF]. Goudenhooft et al. (Goudenhooft et al., 2019b) reported a decrease into mechanical properties of single fibres between the not-tilted flax fibres and tilted flax fibres, especially for young plants, with almost no difference between the not-tilted /tilted flax fibres at plant maturity. Among the stem cell types, bast fibres are actors in the plant gravitropic reaction through the adjustability of their cell wall performance (Goudenhooft et al., 2019b), but the implication of other cellular types, such as xylem, has also been demonstrated, with a significant evolution of the xylem cell morphology and structure after the tilt stage [START_REF] Petrova | Cell Wall Layer Induced in Xylem Fibers of Flax Upon Gravistimulation Is Similar to Constitutively Formed Cell Walls of Bast Fibers[END_REF]. At the scale of the flax stem, the focus has often been on the bast fibres, which is the reinforcement element of the flax stem [START_REF] Goudenhooft | Investigation of the Mechanical Properties of Flax Cell Walls during Plant Development: The Relation between Performance and Cell Wall Structure[END_REF][START_REF] Réquilé | Exploring the link between flexural behaviour of hemp and flax stems and fibre stiffness[END_REF]. In addition, substantial work have been done on the bast fibre from the pulling side of the flax stems tilted at the VS, those cells even displayed an increased cell diameter and an enlarged lumen [START_REF] Ibragimova | Development of gravitropic response: unusual behavior of flax phloem G-fibers[END_REF].

Flax polysaccharides arguably represent the main component of the stem in terms of the mass, with cellulose being the major component in both xylem [START_REF] Beaugrand | Multi-scale analysis of the structure and mechanical performance of woody hemp core and the dependence on the sampling location[END_REF][START_REF] Nuez | The potential of flax shives as reinforcements for injection moulded polypropylene composites[END_REF] and bast fibres [START_REF] Bourmaud | Main criteria of sustainable natural fibre for efficient unidirectional biocomposites[END_REF]. For instance, the stem epidermis covered by a cuticle, the cambial layer and the primary and secondary xylems have been less documented [START_REF] Lion | BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants[END_REF].

In the flax stem, some biochemical compounds are naturally fluorescent, including aromatic substances or compounds containing conjugated double bonds, and this property can be used for detection via fluorescence imaging techniques. For example, lignins and hydroxycinnamate exhibit intense fluorescence in the UV and visible regions.

In proteins, the dominant fluorophore is the indole group of tryptophan, which absorbs near 280 nm and emits approximately 340 nm. In particular, synchrotron radiation provides deep-UV illumination (DUV), enabling multipurpose molecular fluorescence identification [START_REF] Giuliani | DISCO: a lowenergy multipurpose beamline at synchrotron SOLEIL[END_REF]. Using a synchrotron source, a spatial resolution compatible with the sizes of the cell and cell wall (10-50 µm and 1-5 µm, respectively) can be attained with a good signal-to-noise ratio [START_REF] Allouche | Coupling hyperspectral image data having different spatial resolutions by extending multivariate inter-battery Tucker analysis[END_REF]. The excitation at 275 nm coupled with an emission in the range 327-353 nm makes it possible to visualize the proteins thanks to the natural fluorescence of tryptophan and tyrosine, which they harbour in their amino acid sequence [START_REF] Jamme | 3D Imaging of Enzymes Working in Situ[END_REF]. Using the same excitation wavelength, phenolic compounds exhibit natural fluorescence and can be detected at the cell wall scale in lignocellulosic plants at an emission over 380 nm (Devaux et al., 2018a).

Because of fluorescence emission bandpass filters, hydroxycinnamate acids can be partially distinguished from lignin-polymerized phenolic components. Indeed, fluorescence emission bandpass filters at 420-460 nm and 480-550 nm can be used to highlight hydroxycinnamate acids and lignin differentially [START_REF] Jamme | Deep UV autofluorescence microscopy for cell biology and tissue histology[END_REF].

Since polysaccharides are not autofluorescent, mid-infrared (FTIR) spectroscopy has been used to track polysaccharide compositions. Indeed, FTIR microspectroscopy has proven its value in monitoring hemicellulose metabolism in flax [START_REF] Chabi | A Cell Wall Proteome and Targeted Cell Wall Analyses Provide Novel Information on Hemicellulose Metabolism in Flax[END_REF].

Additional work has highlighted the interest of this approach for flax, examining growthinduced modifications of cells [START_REF] Stewart | Fourier-Transform Infrared Microspectroscopy of Anatomically Different Cells of Flax (Linum usitatissimum) Stems during Development[END_REF]. In this study, Stewart et al. reported the spectra of bast fibre cell walls with a predominance of cellulose absorbance. In addition, this work suggested the presence of acetylated hemicelluloses (xylan and/or glucomannan).

In addition to the polysaccharidic components, protein and phenolic compounds play also a major role on metabolism and defence mechanisms of flax cell walls. Major achievements have been reported regarding flax protein expression [START_REF] Corbin | Role of protein farnesylation events in the ABA-mediated regulation of the Pinoresinol-Lariciresinol Reductase 1 (LuPLR1) gene expression and lignan biosynthesis in flax (Linum usitatissimum L.)[END_REF], as well as genome identification and evolution patterns of the main protein family according to differential regulation [START_REF] Corbin | A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation[END_REF]. In addition, phenolic compounds such as hydroxycinnamates are most often esters linked to polysaccharides and esters or ethers linked to lignin monolignols. These compounds can act as interpolymer coupling agents, for example, between polysaccharides and lignin or polysaccharides and structural proteins containing tyrosine amino acids. Hydroxycinnamates, such as lignans, also play an important role in plant defence [START_REF] Mnich | Phenolic cross-links: building and deconstructing the plant cell wall[END_REF]. An attempt to identify and localize phenolic compounds in cell walls by microscopy was made using a laccase tagged with colloidal gold; this enzyme was able to bind to phenolic compounds [START_REF] Gorshkova | Composition and distribution of cell wall phenolic compounds in flax (Linum usitatissimum L.) stem tissues[END_REF]. The authors revealed several gold particle distribution patterns, with compact labelling in xylem and bast fibres; a large proportion of bast fibre wall phenolics consisted of phenolic acids rather than lignin. Lignin is often described as a 3D phenolic polymer [START_REF] Liao | A sustainable wood biorefinery for low-carbon footprint chemicals production[END_REF]. In flax, lignin is mainly concentrated in xylem, and the bast fibre lignin content is very low. The spatial regulation of constitutive lignin under development or during stress is well documented (Le [START_REF] Roy | Spatial regulation of monolignol biosynthesis and laccase genes control developmental and stress-related lignin in flax[END_REF]. The xylem histological part of the stem was particularly well investigated with an elaborate chemistry strategy coupled with a creative microscopic investigation [START_REF] Lion | BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants[END_REF].

The general basics regarding flax plants, their developmental stages and bast fibre characteristics is address in the literature with some recommended papers (Goudenhooft et al., 2019a;[START_REF] Petrova | AFM analysis reveals polymorphism of purified flax rhamnogalacturonans I of distinct functional types[END_REF], but the study of the impact of stress on the cell wall biochemistry or ultrastructure of other flax stem cell types is sparse.

In the current study, FTIR microspectroscopy was performed on only bast fibres. This choice is motivated by the economic interest of that fibre and because it is well documented in the literature in comparison to the others type of cell.

The purpose of this research is to monitor when, where and with what amplitude the biochemical components in flax varied using FTIR (bast fibre only) and deep UV fluorescence after a gravitropic event. Two conditions were chosen: i) during the normal growth of the flax plant as a control plant and ii) following severe stress, namely, 90° bending, which generates gravitropism. These two growing conditions were combined with the three main stages of flax development (vegetative, fast growth and mature stage), and for each, the proteins, hydroxycinnamate and lignin distributions and relative amounts were studied for the five stem cell types investigated.

Material and methods

Flax stems, sampling protocol and growing conditions

Flax plants (Linum usitatissimum L., Bolchoï, seeds provided by Terre de Lin, France) were studied. This variety is this variety is dedicated to the production of fiber fibre. The protocol followed is described in (Goudenhooft et al., 2019b). Briefly, seeds were sown in pots and cultivated outdoors (Lorient, France) in 2017 at a conventional seeding rate of approximately 1800 seeds/m² [START_REF] Bourmaud | Impact of the seeding rate on flax stem stability and the mechanical properties of elementary fibres[END_REF].

A control (C) batch containing plants that never tilted was used as a reference. Three additional pots were used for the experiments, consisting of pots inclined at approximately 90° at different key stages of plant development (Fig 1).

Figure 1 The first pot was tilted at the beginning of the VS, when plants reached approximately 25 cm, 29 days after sowing and a cumulative temperature of 370°C. The second pot was inclined during the FG period, when plants were approximately 50 cm, 40 days after sowing and 510°C of cumulated temperature. Finally, the third pot was tilted at plant maturity (M), in the present case when plants were approximately 80 cm, 77 days after sowing and when the cumulative temperature reached the expected 1000°C [START_REF] Goudenhooft | Varietal selection of flax over time: Evolution of plant architecture related to influence on the mechanical properties of fibers[END_REF].

At each stage of tilting, some plants were sampled. Immediately after sampling, stem portions were fixed overnight in a solution of 4% v/v formaldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C.

Deep UV Fluorescence imaging at the SOLEIL synchrotron DISCO beamline

The DISCO beamline and the imaging microscope setup have been fully described in [START_REF] Giuliani | DISCO: a lowenergy multipurpose beamline at synchrotron SOLEIL[END_REF] and in Jamme et al. [START_REF] Jamme | Deep UV autofluorescence microscopy for cell biology and tissue histology[END_REF]. The socalled TELEMOS microscope is a modified full-field microscope (Axio Observer Z1, Carl Zeiss GmbH, Germany) coupled to the monochromatized synchrotron beam. The power delivered on the sample at the DISCO beamline imaging branch is limited to a few microwatts (1.1*10-6 W at 280 nm). This value avoids beam photodamage by deep UV absorption. The synchrotron beam aligned and focused on the sample using an Ultrafluar 40x (NA 0.6) objective provides a field of view of 250*250 µm² with a pixel size of 244 nm.

The microscope is equipped with a 16-bit back-illuminated CCD camera (Pixis BUV, Princeton Instrument, USA) that allows 65536 grey levels to code the fluorescence intensity and an image size of 1024 x 1024 pixels. The acquisition of images is driven by the ImageJ plugin µManager [START_REF] Edelstein | Computer control of microscopes using µManager[END_REF].

The excitation wavelength was set at 275 nm. A dichroic mirror at 300 nm and three bandpass filters were used for the acquisition of multispectral images. The emission filter at 327-353 nm selected the fluorescence of tryptophan and tyrosine found in the proteins, and the emission filter at 420-460 nm made it possible to acquire images of phenolic compounds corresponding to both the hydroxycinnamic acids and lignin. The emission filter at 480-550 nm was retained as a more specific lignin [START_REF] Frédéric | Deep UV autofluorescence microscopy for cell biology and tissue histology[END_REF] however more recent investigations has shown that other phenolics compounds like flavonol, condensates tannins, anthocyanins can fluorescence in the same range as lignin do (Vidot et al. 2019). Therefore, the signal collected between 480-550 nm cannot guarantee the solely the lignin emission. Image acquisition was performed at room temperature, and the acquisition time was set to 10 s for the three filters. Finally, visible images in transmission mode were acquired in the sequence of multispectral acquisition. The acquisition time was set to 100 ms for the transmission images.

The reference images for background and illumination heterogeneities were acquired once per day, and the run of the entire acquisition covered 3 days. The background images corresponded to the signal recorded by the camera without any illumination and any sample. Using the current camera, the background level was approximately 700 and depended on the filter, acquisition time and location in the image. One background image per bandpass filter was acquired by setting the acquisition time accordingly to 10 s. The illumination field depends on the synchrotron beam alignment and the focal plane. The images of the illumination were acquired using a luminescent reference compound (Nd-YAG crystal) using only the dichroic mirror without any filter and setting the acquisition time to 250 ms. A Z-stack of 101 images was acquired with a 2 µm step between each image.

Flax stem whole sections with a 45 µm thickness were obtained using a HM650V vibratome (Microm International GmbH, Waldorf, Germany) and were deposited on a 1" diameter quartz coverslip (R525000, Esco Optics). Just before the acquisition, the sections were mounted in distilled water and sandwiched with a second circular quartz coverslip. A 10× (NA 0.2) Ultrafluar Zeiss (Carl Zeiss GmbH, Germany) lens was used to provide a field of view of 1.116 × 1.116 µm² with a pixel size of 1.092 μm. Stem sections were approximately 3-4 mm large, and a single image was not sufficient to observe the whole section. A series of images were acquired to cover the whole surface of the section after defining a region of interest in the centre of the images that corresponded to the region illuminated by the synchrotron beam. Depending on the section, between 20 and 30 individual images were acquired to scan the whole section.

Fluorescence image preprocessing and analysis

The fluorescence intensity around the stem section was examined for the 5 cell types including the epidermis (ep), the bast fibre (bf), the cambium (ca), the primary (px) or the Fluorescence images were preprocessed in Devaux et al. (Devaux et al., 2018b) using the principles described in Tomazevic work [START_REF] Tomazevic | Comparative evaluation of retrospective shading correction methods[END_REF] for shading correction. The goal was to remove the camera background and compensate for the illumination inhomogeneity using eq 1:

IMC λ = λ λ eq (1)
where IM(λ) is the raw image acquired using filter λ, BKG(λ) is the additive background, ILL(z) is the z th image of the illumination reference Z-stack and IMC(λ) is the preprocessed image. In the present work, the z th illumination image was obtained by 1) calculating an illumination image as the maximum intensity of all individual images of the section and 2) selecting the z th image ILL(z) in the illumination reference Z-stack as the most correlated to the estimated illumination image. The main steps of image preprocessing are illustrated in the Supplementary Data section.

Supplementary Data section

Images of the whole section were finally built for the three emission filters by merging the preprocessed images into three mosaic images, called composite images, using the information provided by the moving stage. The intensities at the stitching interfaces were set to the maxima between the two adjacent individual images.

Fluorescence image analysis included image observation and fluorescence intensity measurement for each given cell type.

Table 1 False colour images were built from the three emission filters, as described in Table 1. In the resulting colour images, the regions rich in proteins are identified as mainly blue regions, the regions rich in hydroxycinnamates are identified as green regions, and the regions rich in the assumed lignin signal are identified with a colour varying between yellow and red (see Localized spectra were acquired by mid-infrared microspectroscopy using a Tensor 27 (Bruker Optics) spectrometer equipped with a Hyperion 2000 microscope (Bruker Optics).

Visible images were obtained using a Sony camera (Exwave HAD, SSC-DC80P). A 36x objective was selected, and depending on the cell sizes and stage of development, an aperture of 400 to 1600 µm² was set. Infrared spectra were collected in the 4000-700 cm - 1 range at a resolution of 8 cm -1 . Each spectrum summed 700 scans for the background and 500 scans for the samples.

All spectra were baseline corrected and normalized using the acquisition software OPUS7.5 (Bruker optics). The spectra were cropped to the 2000-700 cm -1 range, highlighting the polysaccharide region of interest for the bast fibre components. Norris gap second-derivative spectra were assessed using Unscrambler software (version 10.1, Camo). The second-derivative spectra were thereafter multiplied by (-1). Then, the thick bast fibre wall is arranged in bundles (bf), followed by an area of several layers of small and active cells where the cambial area is located. The cambial cells are small and full of cytoplasm, it is expected that the fluorescence intensity signal recorded there will mix the fluorescence of the cytosol and the wall. Further on, we can see the xylem stained in red due to its high content of lignin, the xylem divided into (xs) as the youngest secondary xylem, and finally the oldest xylem as (xp) is the primary xylem buildup by the plant. lignin mainly originates from the polymerization of trans-para-coumaryl alcohol [START_REF] Day | La lignification des fibres périphloémiennes du lin (Linum usitatissimum L. ) : approches cytochimique, chimique et moléculaire[END_REF]. Lignins can also be linked to cell wall proteins [START_REF] Monties | Molecular structure and biochemical properties of lignins in relation to possible self-organization of lignin networks[END_REF] and to phenolics, ferulic or p-coumaric acids [START_REF] Scalbert | Ether linkage between phenolic acids and lignin fractions from wheat straw[END_REF]) [START_REF] Monties | Molecular structure and biochemical properties of lignins in relation to possible self-organization of lignin networks[END_REF]. Thus, during the implementation of lignin, its structure can take different configurations related to the different fluorescence responses.

Evolution related to the tilted effect

For the 90° tilted plant, the evolution is not as linear as that during the development stage

For the plant inclined at 90 °, the evolution is more contrasted than during the development phase of the control plants. VS_90 presented more green fluorescence than FG_90 and M_90. More blue fluorescence is seen in FG_90 than in the two other stages.

When the gravitropic response takes place during the VS, the cambial region is more intense than the control (see Fig 3a&d), suggesting greater cambial activitymetabolism with tilting. In the VS, the main difference is a more intense green ring visible in the innermost stem tissue, corresponding to the primary xylem. One hypothesis to explain this modification could be a consequence of a modification of the transport in the plant, probably of the sap after the curving of the stem, which could induce a mobilization or translocation of hydroxycinnamates. It is generally admitted that primary metabolic activity is reflected by the presence of proteins [START_REF] Buchanan | Biochemistry & molecular biology of plants[END_REF]. These proteins are cell effectors, structural proteins, etc. involved in the major processes of living organisms and cells. Arguably, hydroxycinnamate is a secondary metabolite. Their localization and modifications therefore reflect metabolic activity but are not as strong as the primary metabolism of proteins.

The restoration of the plant vertical position also occurred in the FG period ( Finally, no polarisation of the signal in response to tilting is analysed 'by eye' observation.

This comment is mandatory because a polar gravitropic response is possible, visible at the scale of the stem between the opposite and bending sides [START_REF] Ibragimova | Development of gravitropic response: unusual behavior of flax phloem G-fibers[END_REF].

Indeed, it has been reported that some morphological modifications in response to gravitropism can take place in the bast fibre but only when bending takes place at a juvenile plant stage.

This qualitative observation of the signals by eye offers information, but quantitative information would provide more information. Therefore, we measured the fluorescence intensity on the cross sections, as explained later below.

Fluorescence quantitative analysis according to the development stages and tilting conditions

For a quantitative fluorescence analysis, an arbitrary virtual division of the stem section into four quarters, called 1 to 4, was performed, as illustrated in Fig 4 A.

Figure 4 The fluorescence intensity collected for each filter allows quantitative evaluations in the fluorescence count unit.

From those average fluorescence intensity values, an analysis of variance (ANOVA) was performed for each filter (Fig 5 ).

Figure 5 The developmental stage, tilting, cell types and all interactions were found to be significant. Because proteins, hydroxycinnamates and lignin arguably do not fluoresce in the same manner and in the same environment, a higher fluorescence does not necessarily mean higher molar amounts; therefore, it is not possible to compare the

At the cell type scale, evolution according to development stage

To go further in the analysis, we looked at the fluorescence intensity according to the cell types. Due to the economic importance of flax bast fibres, more literature is available regarding their composition [START_REF] Akin | Linen most useful: perspectives on structure, chemistry, and enzymes for retting flax[END_REF][START_REF] Bourmaud | Towards the design of highperformance plant fibre composites[END_REF][START_REF] Bourmaud | Main criteria of sustainable natural fibre for efficient unidirectional biocomposites[END_REF] and evolution during plant growth and in response to stress (Goudenhooft et al., 2019a). In connection with Fig 3, the colour of the bast fibre evolves with maturity; in the FG stage, the blue colour representing mainly proteins is evident with a homogenous colour within the fibre cell wall, indicating probably the achievement of the transformation of the immature Gn wall into the mature G at this stage [START_REF] Gorshkova | The snap point: a transition point in Linum usitatissimum bast fiber development[END_REF]. This qualitative observation is supported by the protein fluorescence peak observed in Fig 6

(see FG-C, 'Bast Fibre Proteins'). The structure of bast fibres has been demonstrated to be progressive, with a first Gn layer composed of cellulose and long galactan chains evolving towards a more structured G layer. In this new and mature layer, the length of galactan chains is reduced under the action of galactosidase enzymes, inducing their entrapment between cellulose microfibrils [START_REF] Gorshkova | Aspen Tension Wood Fibers Contain β-(1→4)-Galactans and Acidic Arabinogalactans Retained by Cellulose Microfibrils in Gelatinous Walls[END_REF] and a more cohesive and stiff [START_REF] Arnould | Better insight into the nano-mechanical properties of flax fibre cell walls[END_REF] structure. Here, protein family identification is not possible since we indifferently detected all tryptophan that belongs to the whole protein class.

However, an increase in AGP and extensin during flax fibre development is more likely [START_REF] Day | Identification of cell wall proteins in the flax (Linum usitatissimum) stem[END_REF]. Finally, the cell walls of the xylem primary seem to display a unique dynamic over developmental stages, as no significant variations could be detected from the VS to the M stage, regardless of the three biochemical components targeted (Fig 6).

At the scale of the cellular type, evolution according to the 90° tilt stress

The epidermis (ep) fluorescence signals of the proteins, hydroxycinnamates and lignin did not seem to change in stems after 90° tilt stress (Fig 6). The gravitropic response may also be difficult to detect in this thin layer due to an important SD that could mask a moderate physiological plant response.

For the cambial area, all 3 biochemical families exhibited a stronger intensity after tilt, regardless of the maturity stage considered. This finding highlighted the impact of gravitropism on the cambial response and the preponderant function of this tissue in the plant reaction.

The xylem secondary cell wall type is affected by the gravitropic response that follows the 90° tilt (Fig 6). Regarding the protein, signal intensities Indeed, the intensities in FG and M stages were significantly higher than those in the controls. The hydroxycinnamate signals were more intense in the VS and the FG and M stages than in the control stems.

The lignin signal follows the same trend but with a smaller contrast between controlled and tilted plants.

The xylem primary cell wall type revealed only an increase in the protein signal at the FG stage. In contrast, for hydroxycinnamate, the FG stage is the only stage unaffected by the 90° tilt stress. Compared to control stems, the lignin signal was higher in both the VS and the M stage, but the FG stage was unaltered.

Finally, the bast fibre after 90° tilt could be expected to be involved in the response of the plant. Tilting is known to disrupt normal fibre thickening and lignification in tension wood fibre walls [START_REF] Ghislain | Diversity in the organisation and lignification of tension wood fibre walls -A review[END_REF]. Here, the fluorescence intensity recorded for proteins does not support a modification of the protein within the cell wall if tilt occurs in the VS or FG stage. The protein signals were unchanged between the control and tilted plants, in accordance with both our visual observations (Fig 3) and with the findings of Goudenhooft et al. [START_REF] Goudenhooft | Investigation of the Mechanical Properties of Flax Cell Walls during Plant Development: The Relation between Performance and Cell Wall Structure[END_REF]. However, a slight protein signal increase was observed if tilt occurred in the M stage. Tilting induces an increase in the hydroxycinnamate signal, and the highest difference between tilting and control plants was observed during the FG stage. For lignin, all tilted plants harbour a slight increase in the fluorescence compared to the control plant, and lignin is a well-known polymer involved in plant stress mechanisms.

FTIR on the bast fibre

To supplement the deep UV fluorescence examination of the 5 cell types, a study was performed by FTIR microspectroscopy to investigate the polysaccharide composition; this method has been successfully performed on wood fibres [START_REF] Gierlinger | In situ FT-IR microscopic study on enzymatic treatment of poplar wood cross-sections[END_REF] and flax mutant plants [START_REF] Dymińska | Spectroscopic characterization of genetically modified flax fibers[END_REF]. We focused on the most targeted cell type of the flax stems in the literature, namely, the bast fibres.

FTIR and the development effect on bast fibre Figure 7 A micro-FTIR investigation was performed on the bast fibre cellular type. The main mass at 1200-700 cm -1 is assigned to polysaccharides, such as cellulose, and heteropolysaccharides, such as hemicelluloses and pectin.

The band at approximately 1460 cm -1 is assigned to the CH2 bending of lipids when associated with the C-H2 stretching bands at approximately 2850 and 2920 cm -1 (not shown). The bands at 1735 and 1245 cm -1 correspond to esters, while the absorption bands at approximately 1640 and 1540 cm -1 are signatures of the presence of proteins.

The 1640 cm -1 band also refers to fibre internal water.

Figure 8 The second-derivative spectra were processed to increase spectral differences in the 1800-700 cm -1 region (Fig 8). Cellulose, the major constituent of flax fibres, is identified by the spectral bands at 898, 995, 1010, 1030, 1060, 1105 and 1160 cm -1 [START_REF] Maréchal | The hydrogen bond network in I <SUB>β</SUB> cellulose as observed by infrared spectrometry[END_REF]. The hemicelluloses, O-acetylated glucomannan, xylan and xyloglucan are also present in flax fibre absorbed in the same spectral region as cellulose but can be revealed by the shoulder at approximately 940 and 815 cm -1 [START_REF] Himmelsbach | FT-IR microspectroscopic imaging of flax (Linum usitatissimum L.) stems[END_REF].

The youngest development stage (Fig 8) differs from the later stages by bands assigned to lipids (1735 and 1460 cm -1 ) and adsorbed water (1640 cm -1 ). The peak at 1010 cm -1 is associated with two shoulders at approximately 1600 and 1430 cm -1 , assigned to carboxylate symmetric and antisymmetric COO -bands, which correspond to residual pectin. Its methylated or acetylated form is also present by bands at 1735 and 1245 cm -1 .

The variation in band intensities assigned to cellulose and hemicelluloses at 1010 and 1050 cm -1 mainly arises from the change in the pectins/cellulose or cellulose conformation ratio. Regarding the non-cellulosic polysaccharides, pectic galactan, as well as their processing enzymatic machinery [START_REF] Goubet | Evidence for several galactan synthases in flax (Linum-Usitassimum L) suspension-cultured cells Plant and[END_REF], are known to be part of the bast fibre and evolve according to cell wall modelling [START_REF] Mikshina | Structural details of pectic galactan from the secondary cell walls of flax (Linum usitatissimum L.) phloem fibres[END_REF], while galactan and and proteins capable of modifying it play a role with cellulose within Gn to G development [START_REF] Gorshkova | Occurrence of cell-specific galactan is coinciding with bast fiber developmental transition in flax[END_REF][START_REF] Roach | Development of cellulosic secondary walls in flax fibers requires betagalactosidase[END_REF].

FTIR and tilt effect on the bast fibre and only if the tilt was at the stage of the G-layer deposition (middle and lower part of stem) [START_REF] Ibragimova | Development of gravitropic response: unusual behavior of flax phloem G-fibers[END_REF]. We hypothesize that this effect is not observed in our report because we may not have sampled the same stem section; for instance, 10 cm long samples were taken 2 cm after the cotyledon versus sampling 1 cm after the cotyledon [START_REF] Ibragimova | Development of gravitropic response: unusual behavior of flax phloem G-fibers[END_REF]. In other words, we may have bypassed the area where this polarity occurred, probably by sampling slightly too far in the stem, even if the stem sampling area was slightly curved.

Conclusion

The After a 90° tilt of the flax stem, generally, the fluorescence intensity was higher in all five cell types. This molecular investigation shows that full histological flax stem organization is involved in the gravitropic response following tilting. Our data support the fact that the gravitropic response is also dependent on the metabolic activity of the plant, and the variations in proteins, hydroxycinnamates and lignin are influenced by the developmental stage when tilt occurs.

For cambium as well as the secondary xylem, the active cells are strongly affected by The three channels superimposed

& gravitropism

Qualitative and quantitative biochemical mapping

Lignin and unidentified (Red)

  secondary (sx) xylem shown in Fig 2f.

Figure 2

 2 Figure 2

  Fig 2). Fluorescence intensities were measured in the images for each filter by manually selecting pixels corresponding to different cell types: epidermis (ep), bast fibres (bf); cambium (ca), secondary xylem (xs) and primary xylem (xp). For each cell type, two sets of pixels were selected from each of the four quarters of the section. The intensities were averaged over pixels, resulting in 5 cell types x 4 quarters x 2 repetitions x 3 filter measures per image. The intensities were compared using variance analysis by considering three experimental factors: development stage, tilting conditions, cell types and their interactions. Image processing and variance analyses were performed using MATLAB 2019a with the commercial 'Statistics and Machine Learning' toolbox' (The MathWorks, France).' Fluorescence profiles were extracted using a home made Matlab software allowing the multispectral analysis of the images. Variance analysis was performed using Matlab anova function. The methods allow to recover the mean values and the standard deviations of the effect tested. 2.4 Fourier transform infrared microspectroscopy -spectral analysis Small cylinders (approximately 5 mm in length) of flax stems from the 3 development stages (see section 2.1 and Fig 1), under normal conditions and under stress with a 90° tilt, were embedded in paraffin (Automate Tissue Tek VIP 3000). Sixteen thick cross sections were obtained using an HM355S microtome from Microm and were collected on ZnS windows. The sections were submitted to a Histochoice clearing agent (Sigma H2779) solution to remove the paraffin.

3.

  Fig 2 shows the fluorescence images of the flax stem cross sections at the young

Figure 3 Fig 3

 33 Figure 3

  Fig 3 b&e). During this step, the cambial region of tilted samples (Fig 3e) also seemed to be more fluorescent than the control samples (Fig 3. d). The protein signal areas and intensities (blue) were more pronounced, with the bast fibres being greener fluorescent (hydroxycinnamate) than the control plants. During the gravitropic reaction (Fig 3 c&f), a much higher fluorescence intensity was observed at M_90 than at the control M_C. Despite the presence of well-mature cell walls, especially in the primary tissues, the cambial activity fluorescence of tilted plants (Fig 3f) was still present but more limited than that of tilted plants at the younger stages, as shown by the reduction in the blue xylem "ring" in contact with the cambium (Fig 3 d&e). Moreover, the area and intensities of the signals coming from hydroxycinnamates are much greater than those from any other samples, proving that mature cells are still able to react to inclinationbending. This reaction most likely affects the lignification process or lignin cellular types such as xylem and hydroxycinnamate, which are precursors of this cell wall component.It seems by eye that mature fibres are not strongly involved in the gravitropic reaction, whereas the xylem secondary part is. In addition, regarding the bast fibre, the signal intensities of M_90 seemed lower than those of tilted plants from previous growth stages and were similar to those of the control M_C plants.

  different components as molar amounts based on their respective signals. If we focus first on the development stage effect, then the average fluorescence intensity for the three emission channels corresponding to proteins (Fig 5a), hydroxycinnamates (Fig 5b) and lignin (Fig 5c) varied between the 3 development stages. The highest intensity for proteins and hydroxycinnamates was observed in the fast growing stage for both the control and 90° tilted stems (FS_C or FS_90), and the lowest intensity was observed in the vegetative stage (VS_C or VS_90°). This trend is different for lignin: an increase in the intensity occurred between stages VS and FG before reaching a plateau. In contrast, the lignin fluorescence seemed equal for the three developmental stages of the 90° tilted stems. If we focus now on the tilt effect, then the gravitropic response systematically induced an increase in the fluorescence intensity in the three channels compared to the control plant stems, by 51, 28 and 23% for proteins, hydroxycinnamate and lignin, respectively. The 90° tilt gives rise to an average increase of 32% of the fluorescence (Fig 5), somehow confirming the qualitative observation (section 3.1, Fig 2). This result suggests a global boost in plant metabolism following lodging stress.

Figure 6 Fig 6

 66 Figure 6

  The cambium fluorescence signals(third line of histogram, Fig 6) for proteins, hydroxycinnamates and lignin show similar trends to the bast fibre, i.e., an increase between the VS and FG stage, followed by a slight decrease in the M stage. The fluorescence intensity in cambium is rather high, and the variations between developmental stages are marked.The secondary xylem (xs) cells demark with the other cell wall types. Indeed, for fluorescence signals attributed to hydroxycinnamates or lignin, there was a significant decrease from the VS to the M stage. The protein fluorescence is higher than that of the other cell types, and signals for proteins fall drastically between the FG and M stages. In contrast, for the signals attributed to hydroxycinnamates and lignins, the decrease is progressive between the VG and M stages. Regarding lignin, a recent report using an elegant tag of lignin specifies the active areas of lignification in the secondary xylem of flax[START_REF] Lion | BLISS: A Bioorthogonal Dual-Labeling Strategy to Unravel Lignification Dynamics in Plants[END_REF]. The authors showed temporal lignification differences among the xylem vessel, ray parenchyma cells and tracheid cells, as well as revealing the polarization at the scale of a single-cell lignification dynamics. In this work, such resolutions could not be achieved, but we observed a slight decline in the fluorescence intensity in stages FG and M compared to the VS (Fig6).

Fig

  Fig 7A illustrates the location of micro-FTIR spectra in a flax stem section. An average

Figure 9

 9 Figure 9

Figure 10 Then

 10 Figure10

  aim of this work was to gain new insights into the dynamics of the cell wall composition in the different cell types of the flax stem during plant development and after a 90° tilt bending stress. The autofluorescence properties of proteins and phenolic compounds when excited at a deep UV (DUV) wavelength accessible to the SOLEIL synchrotron and multichannel fluorescence imaging have made it possible to track proteins and hydroxycinnamates. At contrary, whereas the quantification of the lignin fluorescence by the 480-550 nm filter could not be attained is our case. The specificity of this filter was checked on extracted polymers. In our work, the higher amount of fluorescence observed using this filter in both epidermis or cambium compared to xylem cannot be assigned to lignin. We hypothesis that non-polymeric molecules, unidentified in this work, overlap with the fluorescence of lignin. Further investigation are needs to elucidate whose molecules are concerned. The main output is that there are higher fluorescence signals of the hydroxycinnamate and both 'lignin' and proteins at the FG stage for the five cell types of the stems, without exception. The fluorescence intensities decrease at the M stage.

Figure 1 .

 1 Figures caption

Figure 2 .

 2 Figure 2. Individual channel fluorescence and composite view of the VS_C flax stem

Figure 3 .

 3 Figure 3. Composite fluorescence images of the control plants _C or tilted plants _90,

Figure 4 .

 4 Figure 4. Fluorescence measurement in the cell types. A is the full stem section with the

Figure 5 .

 5 Figure 5. Average values of the fluorescence intensity (in arbitrary units) obtained from

Figure 6 .

 6 Figure 6. Cellular type average fluorescence intensity according to development and tilting conditions. The drop caps indicate whether the values are significantly different from each other or not.

Figure 7 .

 7 Figure 7. FTIR spectrum of bast fibres.

Figure 8 .

 8 Figure 8. Effect of the development stage. The bast fibre second-derivative micro-FTIR

Figure 9 .

 9 Figure 9. Effect of a 90° tilt on the second-derivative micro-FTIR spectra of the flax bast

Figure 10 .

 10 Figure 10. Investigation of the putative polarity in the stem by exploring the fluorescence

Figure 1 .

 1 Figure 1. Scheme of the different steps of the experiment. The top part: a) Schematic representation of the flax stems at different maturity stages. The pots were never inclined and so acted as control (_C) conditions, whereas other pots were tilted for 4 days. Tilting (_90) was performed at three major stages of plant development (VS, FG and M), and tilted samples were collected 4 days after tilting, as depicted in the lower part b). About 10 cm stem portions taken approximately 2 cm above cotyledons were sampled from whole stems and are illustrated by blue rectangles in b). Overall, 6 flax stem conditions were explored (VS_C, VS_90, FG_C, FG_90, M_C and M_90).

Figure 2 .

 2 Figure 2. Individual channel fluorescence and composite view of the VS_C flax stem cross section. a) Fluorescence intensity of the channel of proteins, b) channel of hydroxycinnamate in c) channel of lignin, d) the composite view with the merged a-c channels, in e) a guide to interpret colours and in f) a phloroglucinol staining of the quarter of the stem section to present the 5 cellular components of this study. The field of view is 2.7 x 2.5 mm² for images a to d. In f, the inner stem part to the epidermis is (ep), the bast fibre (bf), the cambium (ca), the xylem -secondary (xs) and the xylemprimary (xp).

Figure 3 .

 3 Figure 3. Composite fluorescence images of the control plants _C or tilted plants _90, magnification 10x. Stem cross section from a control plant (a) and from a tilted plant (d) during the vegetative stage VS. Stem cross section from control (b) and tilted (e) plants during fast growth FG. Stem cross section of mature M plants and control (c) and tilted (f) plants. In b, a caudal pod is visible on the left side of the stem.

Figure 4 .

 4 Figure 4. Fluorescence measurement in the cell types. A is the full stem section with the 4 sampling areas (labelled 1, 2, 3 and 4). The different cell types selected for fluorescence intensity quantification are indicated. B focuses on area 1 to illustrate the manual selection of pixels in the different cell types; the red lines go from the outer part to the inner stem part to the epidermis (ep), the bast fibre (bf), the cambium (ca), the xylem -secondary (xs) and the xylem -primary (xp). A zoom is provided for each vignette.

Figure 5 .

 5 Figure 5. Average values of the fluorescence intensity (in arbitrary units) obtained from the ANOVA development stages × conditions. The drop caps indicate whether the values are significantly different from each other or not.

Figure 6 .

 6 Figure 6. Cellular type average fluorescence intensity according to development and tilting conditions. The drop caps indicate whether the values are significantly different from each other or not.

Figure 7 .

 7 Figure 7. FTIR spectrum of bast fibres. In a), the blue dots indicate the location of the bast fibre local spectra collected around the flax stem samples. In b), the absorption bands for the main components in bast fibre are indicated.

Figure 8 .

 8 Figure 8. Effect of the development stage. The bast fibre second-derivative micro-FTIR spectral signals of control plants are superimposed. Red arrows indicate the bands of interest for the study.

Figure 9 .

 9 Figure 9. Effect of a 90° tilt on the second-derivative micro-FTIR spectra of the flax bast fibre. Red arrows indicate the bands of interest for the study.

Figure 10 .

 10 Figure 10. Investigation of the putative polarity in the stem by exploring the fluorescence of control versus tilted stems in bast fibres. Fig 10a) shows the arbitrary positioning of the fluorescence measurements around the stem section. Fig 10b) shows the location of the pixel fluorescence measurement in the bast fibre for M_C (square) and M_90 (lozenge). Fig 10c) shows the fluorescence intensity measured and expressed linearly for the tilted/untreated plants.

Table caption Table 1 .

 caption1 Building colour images from the three emission filters. The same maximum intensity values were set for all images.

Acknowledgements

The authors are grateful to Marc Lahaye and Kevin Vidot for fruitful discussions. The authors acknowledge the financial support from SOLEIL for VV, CD and FC regarding Proposal 20171187. The authors also thank the French national research Network GDR 3710 INRAE/CNRS SYMBIOSE -Synthons et matériaux biosourcés for its support and the ANS μReshape' funding support received from the TRANSFORM department of INRAE. This research was also funded by the INTERREG VA FCE Program, FLOWER project, Grant Number 23.


M Chemkhi
email: mahdi.chemkhi@epf.fr

J Marae Djouda

M A Bouaziz

J Kauffmann

F Hild

D Retraint

Effects of Mechanical Post-Treatments on Additive Manufactured 17-4PH Stainless Steel Produced by Bound Powder Extrusion

Keywords: 17-4PH SS, Bound Powder Extrusion, Post-treatment, SMAT, Roughness, Residual stresses

Bound Powder Extrusion (BPE) is increasingly gaining popularity and makes metal additive manufacturing easier and more accessible for industrial production. The Markforged-Metal X technology with debinding and sintering, also called Atomic Diffusion Additive Manufacturing (ADAM), is a newcomer in the field of extrusion additive manufacturing. The main objective of this work is to investigate the mechanical properties of 17-4PH stainless steel samples produced by ADAM. Moreover, the effects of a mechanical post-treatment (SMAT) on surface and mechanical properties are also analyzed. The macroscopic tensile response and superficial residual stresses of as-fabricated and SMATed samples are compared.

Introduction

Initially, additive manufacturing (AM) was mainly developed with polymers. The most widespread additive manufacturing technology is material extrusion (with a wide range of materials), such as fused filament fabrication (FFF), also known as fused deposition modeling (FDM) [START_REF] Gao | Fused filament fabrication of polymer materials: A review of interlayer bond[END_REF]. Conversely, the powder bed-based process is the predominant technology for Metal Additive Manufacturing (MAM), such as Laser Powder Bed Fusion (L-PBF), also known as Selective Laser Melting (SLM) [START_REF] Nagarajan | Development of Micro Selective Laser Melting : The State of the Art and Future Perspectives[END_REF]. Over the last decades, AM technologies are continuously being developed [START_REF] Ngo | Additive manufacturing ( 3D printing ): A review of materials , methods , applications and challenges[END_REF] and new applications are currently emerging especially through the development of MAM. In addition, according to the Global Metal Additive Manufacturing Market 2020-2024 [START_REF] Technavio | Global Metal Additive Manufacturing Market 2020-2024[END_REF], the interest of industries in many sectors (e.g., aeronautics, automotive, medical, energy) on MAM is growing exponentially. Such processes may be divided into two main categories, namely, direct and indirect metal additive manufacturing [START_REF] Pragana | Hybrid metal additive manufacturing : A state -of -theart review[END_REF]. The first category is based on powder fusing techniques, such as Laser Powder Bed Fusion (L-PBF) and Direct Energy Deposition (DED). The second one is combining printing with debinding and sintering such as Material Extrusion [START_REF] Thompson | Fused filament fabrication, debinding and sintering as a low cost additive manufacturing method of 316L stainless steel[END_REF] or Metal Binder Jetting [START_REF] Ziaee | Binder jetting : A review of process , materials , and methods[END_REF]. Each method has its own advantages and drawbacks [START_REF] Bikas | Additive manufacturing methods and modelling approaches : a critical review[END_REF]. On the one hand, common to both LPBF and DED processes, thermal gradients are due to the solidification of each layers [START_REF] Casati | Microstructure and Fracture Behavior of 316L Austenitic Stainless Steel Produced by Selective Laser Melting[END_REF] in direct MAM, which result in anisotropic properties and residual stresses based on the build direction [START_REF] Michailidis | Comparison of Additively Manufactured vs . Conventional Maraging Steel Conference , after France Maraging Comparison of Additively Manufactured Steel in Corrosion-Fatigue Performance various surface treatments in Corrosion-Fati[END_REF]. On the other hand, indirect MAM is increasing in popularity as an alternative non-fusion-based method [START_REF] Mun | Indirect additive manufacturing based casting of a periodic 3D cellular metal -Flow simulation of molten aluminum alloy[END_REF], in which the powder is lightly bonded as a powder bed (Metal Binder Jetting) or as filaments (Material Extrusion). In this case, sintering via indirect AM induces a relatively uniform temperature profile throughout the part compared to direct AM, which may reduce the anisotropy of the obtained material properties.

Moreover, the needed equipment for direct metal AM processes is very costly to purchase and operate [START_REF] Thompson | Fused filament fabrication, debinding and sintering as a low cost additive manufacturing method of 316L stainless steel[END_REF]. The focus of this article is on the second method, and presents a recently developed indirect additive manufacturing process "Metal X" by Markforged Inc. [12], which combines metal injection molding (MIM) and fused filament fabrication (FFF) technologies into Bound Powder Extrusion (BPE) [START_REF] Galati | Analysis of density, roughness, and accuracy of the atomic diffusion additive manufacturing (ADAM) process for metal parts[END_REF]. This process, which is called Atomic Diffusion Additive Manufacturing (ADAM), provides enhanced design freedom and simplified solutions compared to other direct AM processes.

The first metal powder introduced by Markforged is made of 17-4PH martensitic stainless steel. It is one of the most popular materials thanks to its high-strength and high corrosionresistance [START_REF] Lashgari | Microstructure , Tribological Properties and Corrosion Behaviour of Additively Manufactured 17-4PH Stainless Steel : E ff ects of Scanning Pattern , Build Orientation, and Single vs . Double scan[END_REF]. However, there are limited data on final properties that can be expected, as well as data related to residual stress distributions and their impact on the mechanical properties of the processed parts. Hence, the goal of this study is to investigate the mechanical properties of 17-4PH parts made with the ADAM technology. In addition, since surface features affect the behavior of metallic materials, a mechanical post-treatment (i.e., surface mechanical attrition [START_REF] Portella | Materials Characterization In fl uence of Surface Mechanical Attrition Treatment ( SMAT ) post-treatment on microstructural , mechanical and tensile behaviour of additive manufactured AISI 316L[END_REF]) was performed in order to enhance the surface characteristics and also improve the mechanical properties of printed parts [START_REF] Maleki | Surface post-treatments for metal additive manufacturing : Progress , challenges , and opportunities[END_REF].

Material and methods

Additive manufacturing

The Metal X process consists of four basic steps viz. design, printing, debinding and sintering:

1. The first step converts the virtual model into a series of printable layers using the slicer code "Eiger," and transfers that information to the Metal X printer. 2. Then, printing consists in selectively extruding the fused filament through a nozzle to obtain the "green" part of 17-4PH metal powder bound in a polymer matrix. 3. Next comes debinding with the immersion of this green part in a specialized solvent that dissolves the primary binder to obtain the "brown" part. 4. After solvent debinding all parts were dried before the thermal debinding using the Sinter-1 of the Metal X system for complete removal of the remaining binder. The final step is to sinter that part in a high temperature horizontal tube furnace to obtain a relatively dense metallic form.

As PBF, SLM imposes some restrictions on the 3D model and requires an escape hole for unused powder. However, for the ADAM technology, as the powder burns away during the sintering process, it allows for the fabrication of complex and enclosed internal structures to obtain light parts without sacrificing the part strength (since the powder is contained in a polymer binder, which is removed after printing completion). Then, the polymer eliminates the toxicity and flammability inherent to powders and makes 3D MAM more accessible and faster while reducing manufacturing costs [12]. In this work, the printing parameters were adjusted to produce mechanically stable and dense metallic parts (Table 1). As illustrated in Fig. 1(a), the filling strategy adopted herein is 45° plus 90° with interval rotating. The geometry presented in Fig. 1(b) corresponds to a notched specimen loaded in tension in accordance with the ASTM E1820 [START_REF] Astm Standard | Standard Test Method for Measurement of Fracture Toughness[END_REF] standard. This type of geometry was selected to study the fracture properties of such materials. Preliminary analyses will be discussed hereafter.

Mechanical Surface treatments details

Surface Mechanical Attrition Treatment (SMAT) is based on the vibration of spherical balls using a high frequency ultrasonic generator [START_REF] Eyzat | Characterization and Mechanical Properties of SLM Ti-6Al-4V subjected to surface mechanical post-treatment[END_REF]. SMAT induces strain hardening on the surface induced by plastic deformation via multidirectional and random shot impacts [START_REF] Lu | Nanostructured surface layer on metallic materials induced by surface mechanical attrition treatment[END_REF]. The principle of SMAT is illustrated in Fig. 2. Only few investigations have been performed on similar mechanical surface treatments of manufactured metallic materials such as titanium alloys [START_REF] Eyzat | Characterization and Mechanical Properties of SLM Ti-6Al-4V subjected to surface mechanical post-treatment[END_REF] or 316L stainless steels [START_REF] Sun | Surface finish and properties enhancement of selective laser melted 316Lstainless steel by surface mechanical attrition treatment[END_REF]. They show that SMAT may significantly affect the microstructure of treated MAM alloys, increase the microhardness, reduce the roughness, improve their mechanical properties [START_REF] Portella | Materials Characterization In fl uence of Surface Mechanical Attrition Treatment ( SMAT ) post-treatment on microstructural , mechanical and tensile behaviour of additive manufactured AISI 316L[END_REF], induce compressive residual stresses [START_REF] Portella | Residual Stresses Analysis in AISI 316L Processed by Selective Laser Melting (SLM) Treated by Mechanical Post-Processing Treatments[END_REF], and increase the fatigue resistance [START_REF] Yan | Fatigue strength improvement of selective laser melted Ti6Al4V using ultrasonic surface mechanical attrition[END_REF]. SMAT method may also reduce anisotropy on the surface of MAM samples by inducing high-strain-rate plastic deformation in multiple directions.

The efficiency of such deformation-based process depends on several parameters such as the size and material of the balls, the processing time, frequency, and power of the generator. SMAT was applied to each face of as-fabricated specimens. The SMAT parameters reported in Table 2 were selected based on previous experiments [START_REF] Chemkhi | Effect of SMAT Parameters on Microstructural Features using DOE Technique[END_REF].

Material characterization

The surface roughness of the As-Fabricated (AF) samples was measured by surface profilometry using a Surtronic 3+ equipment from Taylor Hobson. The mechanical properties of AF and SMATed samples were evaluated via tensile tests performed at room temperature using an Instron testing machine. The crosshead velocity was set to 3.5 µm/s.

The samples were also characterized via X-ray diffraction (Seifert PTS-3000) using Cr Kα radiation. The superficial residual stresses were evaluated using the sin²ψ method.

Results and discussions

Surface roughness and topography

The surface of an additively manufactured part is frequently rough and is not suitable for many engineering applications where surface contact [START_REF] Kc | Tribological behavior of 17 -4 PH stainless steel fabricated by traditional manufacturing and laser-based additive manufacturing methods[END_REF], friction [START_REF] Sun | Surface finish and properties enhancement of selective laser melted 316Lstainless steel by surface mechanical attrition treatment[END_REF], and especially fatigue phenomena are significant [START_REF] Nezhadfar | Fatigue behavior of additively manufactured 17-4 PH stainless steel : Synergistic effects of surface roughness and heat treatment[END_REF]. Rough surface profiles may induce stress concentrations and thus become sites for crack initiation. In order to have some indication on how SMAT affects roughness and the surface topography of AF-samples, roughness measurements [START_REF] Gadelmawla | Roughness parameters[END_REF] were conducted on 8 different locations in the samples (Fig. 3).

In accordance with the ISO 4287 [START_REF]Geometrical Product Specifications (GPS) -Surface texture: Profile method -Terms, definitions and surface texture parameters[END_REF] standard, the measured roughness parameters Ra and Rt are reported in Fig. 3(a). The surface topography of the specimens is compared in Fig. 3(b). The surface of AF-samples is rough with many peaks as high as 30 μm and valleys as deep as 20 μm. The average Ra value is about 4 μm, and Rt is about 33 μm. Both Ra and Rt were reduced by more than 90% after SMAT. One interesting feature of Fig. 3(b) is that after SMAT, most of the roughness peaks were removed or flattened such that the surface became smoother with some of the original valleys remaining.

Superficial residual stress

It is known that compressive residual stresses are favorable to hinder crack initiation [START_REF] Coules | The effects of residual stress on elastic-plastic fracture propagation and stability[END_REF]. To understand the influence of SMAT on the level of superficial residual stresses of AF samples, both sets were analyzed via XRD. The AF-samples exhibit tensile stresses up to 500 MPa. After SMAT, compressive residual stresses down to -700 MPa were measured.

Tensile test

In this section, the mechanical behavior of 17-4PH samples before and after SMAT is investigated. Tensile tests were performed with the same testing conditions up to specimen failure [START_REF] Bouaziz | Microscale mechanical characterization of 17-4PH stainless steel Microscale fabricated by by Atomic Atomic Diffusion Diffusion Additive Additive Manufacturing (ADAM)[END_REF]. The tensile data reported in Fig. 4 were carried out to assess basic properties. As SMAT induces plastic deformation, it changes the thickness of treated samples and given that the specimens were notched, the net section stress is plotted as a function of average engineering strain. The responses are very close for each category of samples with a slight shift. The final elongations for AF samples are 7.4 %, 7.8 % and 8.9 %, and 6.6 %, 7.1 % and 8.4 % for SMATed samples. The net section stress calculated for equal engineering strain is higher for SMATed samples all along the test. This trend is also true for the ultimate strength, which reaches 774 MPa for SMATed samples while it does not exceed 742 MPa for AF samples. These experimental results show that SMAT improves the tensile strength and slightly reduces the final elongation of 17-4PH stainless steel obtained via ADAM. All these observations are significant enough to serve as basis for further comparison between tests.

Conclusion

In this paper, different characteristics of 17-4PH stainless steel samples fabricated via Bound Powder Extrusion (BPE) using the Markforged Metal X technology were studied. SMAT was performed with the aim of improving surface properties. The average surface roughness of SMATed samples decreased by about 90%.

Superficial residual stresses were evaluated before and after the mechanical treatment via XRD. SMAT significantly changed initial tensile stresses (up to 500 MPa) into high compressive stresses (down to -700 MPa), which are beneficial for the lifetime of such parts.

Last, tensile tests were carried out on notched samples in order to investigate the effects of mechanical post-treatment on the ultimate strength and elongation to failure. The former was higher for SMATed samples. Conversely, the elongation to failure was slightly higher for AF-samples.

Further experiments are currently under way in order to evaluate the relationship between printing parameters (e.g., number of wall layers, layer height) and resulting sample properties (e.g., dimensional accuracy, mechanical properties).

The sample geometry selected herein will also allow fracture mechanisms to be investigated thanks to full-field measurements (via digital image correlation). When compared with data obtained on un-notched samples, the notch sensitivity of such materials will be evaluated.

Fig. 1 .

 1 Fig. 1. (a) Schematic illustration of filling strategies. (b) Shape and dimensions of the tensile samples with 1.5 mm thickness.

Fig. 2 .

 2 Fig. 2. Illustration of SMAT. (a) Samples and balls placed in the treatment chamber. (b) Vibrating part.

Fig. 3 .

 3 Fig. 3. (a) Surface roughness parameters. (b) Surface profiles of As-Fabricated and SMATed samples.

Fig. 4 .

 4 Fig. 4. Macroscopic stress/strain curves of As-Fabricated and SMATed samples.

Table 1 .

 1 AM processing parameters used in this work.

	Steps	Processing parameters	As-Fabricated Samples
		Filament Material	17-4PH stainless steel
		Metal Temperature	218° ±2 °C
		Infill Pattern	Solid Fill
	Design /	Type of filling	45° plus 90° with interval rotating
	Printing	Raft	Printing with no raft
		Wall layers (contours)	2 walls
		layers height	50 µm
		Print bed	Heated, vacuum-sealed print sheet
		Solvent type	Opteon SF-79
	Debinding	Wash time	12 h
		Dry time	4 h
	Sintering	Peak internal temp. Duration	1300° C 27 h

Table 2 .

 2 Mechanical post-treatment conditions.

	Post-treatment parameters	SMAT High
	Ball	100Cr6 steel
	Ball diameter	3 mm
	Processing time	30 min
	Power and frequency of the generator	30% and 20 kHz

Acknowledgements

The authors wish to thank the FabAdd Platform of EPF Troyes for manufacturing the samples. The 'Troyes Champagne Métropole' and the Aube Department Council (France) are also acknowledged for their financial support of Markforged-Metal X System.