Bofang Yan

Marie-Pierre Isaure

Sandra Mounicou

Hiram Castillo-Michel

Wout De Nolf

Christophe Nguyen

Jean-Yves Cornu

Cadmium distribution in mature durum wheat grains using dissection, laser ablation-ICP-MS and synchrotron techniques

Keywords: Cadmium, durum wheat grain, LA-ICP-MS, µXRF, µXANES

Understanding how essential and toxic elements are distributed in cereal grains is a key to improving the nutritional quality of cereal-based products. The main objective of this work was to characterize the distribution of Cd and of nutrients (notably Cu, Fe, Mn, P, S and Zn) in the durum wheat grain. Laser ablation inductively coupled mass spectrometry and synchrotron micro X-ray fluorescence were used for microscale mapping of Cd and nutrients. A dissection approach was used to quantitatively assess the distribution of Cd and nutrients among grain tissues. Micro X-ray absorption near-edge spectroscopy was used to identify the Cd chemical environment in the crease. Cadmium distribution was characterized by strong accumulation in the crease and by non-negligible dissemination in the endosperm. Inside the crease, Cd accumulated most in the pigment strand where it was mainly associated with sulfur ligands. High-resolution maps highlighted very specific accumulation areas of some nutrients in the germ, for instance Mo in the root cortex primordia and Cu in the scutellum. Cadmium loading into the grain appears to be highly restricted. In the grain, Cd co-localized with several nutrients, notably Mn and Zn, which challenges the idea of selectively removing Cd-enriched fractions by dedicated milling process.

Main finding of the work

This work provides the first high-resolution maps of Cd distribution in durum wheat grain and, thereby, adds to our understanding of the processes involved in Cd loading and storage in cereal seeds.

Introduction

Cadmium (Cd) is a heavy metal contaminant whose toxicity to humans has long been recognized. The European Food Safety Authority (EFSA) established a provisional tolerable weekly intake for Cd of 2.5 μg kg -1 body weight based on the long-term harmful effects of low doses of Cd on human health [START_REF] Efsa | EFSA (European Food Safety Authority) Statement on tolerable weekly intake for cadmium[END_REF]. Though the average dietary exposure is below this level in the European population, some subgroups including children, young women and vegetarians are more likely to frequently exceed it [START_REF] Clemens | Plant science: the key to preventing slow cadmium poisoning[END_REF]. In Europe, almost one third of dietary exposure to Cd is via consumption of cereals [START_REF] Efsa | Cadmium dietary exposure in the European population[END_REF]. Durum wheat has a greater tendency to accumulate Cd in grains than common wheat and other cereals [START_REF] Mclaughlin | Cadmium in soils and plants[END_REF][START_REF] Grant | Selection and breeding of plant cultivars to minimize cadmium accumulation[END_REF]. The cadmium concentration is often above 0.1 mg kg -1 in durum wheat grains and can sometimes exceed the regulation limit of 0.2 mg kg -1 set by the European Union [START_REF] Ec | Commission Regulation (EC) No 629/2008 of 2 July 2008 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs[END_REF]. Therefore, particular attention should be paid to reducing the level of Cd in durum wheat-based products (e.g. semolina, pasta). Substantial efforts have already been made to understand how Cd is taken up, transported, and allocated to the grains in cereal crops [START_REF] Clemens | Toxic heavy metal and metalloid accumulation in crop plants and foods[END_REF][START_REF] Maccaferri | Durum wheat genome highlights past domestication signatures and future improvement targets[END_REF]). However, little information is available on how Cd is distributed within the grain, even though the differential accumulation of an element among grain tissues may mirror its internal loading, storage and functions within the grain [START_REF] Lombi | Speciation and distribution of arsenic and localization of nutrients in rice grains[END_REF]. For instance, the transfer of zinc (Zn) from maternal to filial tissues was proposed as the main barrier to Zn loading into wheat grains, based on the much lower concentration of Zn measured in the endosperm than in the vascular bundles in the crease [START_REF] Wang | Stable isotope labelling and zinc distribution in grains studied by laser ablation ICP-MS in an ear culture system reveals zinc transport barriers during grain filling in wheat[END_REF]). In the same way, the involvement of sulfur (S) complexes in the loading of iron (Fe) into the grain was proposed based on the co-localization of these two elements in the nucellar projection [START_REF] Singh | Pattern of iron distribution in maternal and filial tissues in wheat grains with contrasting levels of iron[END_REF]. A clear picture of how Cd is distributed among tissues and how it is colocalized with nutrients in the grain is thus a key to developing agronomic strategies to improve the food safety of durum-wheat based products. Among nutrients, those to primarily focus on are Fe, Mn and Zn as they share transporters and loading pathways with Cd, and S since Cd has a high affinity for thiol groups and is often bound to Scontaining ligands in plant tissues [START_REF] Clemens | Plant science: the key to preventing slow cadmium poisoning[END_REF].

Significant advances in imaging techniques make it possible to obtain high definition elemental maps of wheat grains [START_REF] Lombi | In situ analysis of metal(loid)s in plants: State of the art and artefacts[END_REF]. For instance, synchrotron micro X-ray fluorescence (µXRF) has been used to investigate the distribution of P, S, Mn, Fe, Cu and Zn in bread wheat [START_REF] Singh | Pattern of iron distribution in maternal and filial tissues in wheat grains with contrasting levels of iron[END_REF][START_REF] Ajiboye | X-ray fluorescence microscopy of zinc localization in wheat grains biofortified through foliar zinc applications at different growth stages under field conditions[END_REF][START_REF] Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF]. Laser ablation inductively coupled mass spectrometry (LA-ICP-MS) can also be used to identify the distribution of metals. This technique has been widely used in recent years to map trace elements such as As, Cu, Fe, Mn, Ni, Pb and Zn in grains of bread wheat [START_REF] Lombi | Speciation and distribution of arsenic and localization of nutrients in rice grains[END_REF][START_REF] Wu | Diverse accumulation and distribution of nutrient elements in developing wheat grain studied by laser ablation inductively coupled plasma mass spectrometry imaging[END_REF][START_REF] Van Malderen | Imaging the 3D trace metal and metalloid distribution in mature wheat and rye grains via laser ablation-ICP-mass spectrometry and micro-X-ray fluorescence spectrometry[END_REF], sunflower [START_REF] Gds | A quantitative approach for Cd, Cu, Fe and Mn through laser ablation imaging for evaluating the translocation and accumulation of metals in sunflower seeds[END_REF], maize [START_REF] Imran | Zinc distribution and localization in primed maize seeds and its translocation during early seedling development[END_REF] as well as in cocoa beans [START_REF] Thyssen | Bioimaging of the elemental distribution in cocoa beans by means of LA-ICP-TQMS[END_REF]. The chemical analysis of dissected tissues is another way to investigate the distribution of elements in plant grains [START_REF] Pieczonka | Distribution of cadmium, copper, and zinc in the caryopsis of wheat (Triticum aestivum L.)[END_REF][START_REF] Kutman | Improved nitrogen status enhances zinc and iron concentrations both in the whole grain and the endosperm fraction of wheat[END_REF][START_REF] Detterbeck | Spatially resolved analysis of variation in barley (Hordeum vulgare) grain micronutrient accumulation[END_REF]. Chemical analysis provides quantitative information at the scale of the whole grain whereas imaging can only show the distribution of elements in a section of the grain.

Only a few studies have attempted to map the distribution of Cd in grains. This is probably because the level of Cd accumulation in plant grains is usually too low to be accurately quantified by imaging [START_REF] Clemens | Toxic heavy metal and metalloid accumulation in crop plants and foods[END_REF]. [START_REF] Van Malderen | Imaging the 3D trace metal and metalloid distribution in mature wheat and rye grains via laser ablation-ICP-mass spectrometry and micro-X-ray fluorescence spectrometry[END_REF] tried to map Cd in bread wheat grain with a Cd concentration of 0.05 µg g -1 but the signal intensity of Cd in some parts fell to the background level, preventing the precise delimitation of areas in which Cd accumulated. The only informative maps of Cd distribution were imaged on Cd-contaminated rice grains [START_REF] Basnet | Elemental bioimaging of tissue level trace metal distributions in rice seeds (Oryza sativa L.) from a mining area in China[END_REF][START_REF] Yan | Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies[END_REF]) and in sunflower [START_REF] Gds | A quantitative approach for Cd, Cu, Fe and Mn through laser ablation imaging for evaluating the translocation and accumulation of metals in sunflower seeds[END_REF]. In rice, [START_REF] Basnet | Elemental bioimaging of tissue level trace metal distributions in rice seeds (Oryza sativa L.) from a mining area in China[END_REF] observed that Cd was distributed throughout the grain whereas, in sunflower, Pessôa et al. (2017) observed that Cd preferentially accumulated in the cotyledons. Today, a high-resolution map of Cd distribution in durum wheat grain is still lacking.

In the present study, LA-ICP-MS was set up to produce images of the localization of Cd and of six mineral nutrients (Cu, K, Mn, Mo, P and Zn) in transversal and longitudinal sections of mature durum wheat grains (including the embryo) at the micrometer scale spatial resolution. Synchrotron µXRF and micro X-ray absorption near-edge spectroscopy (µXANES) were also used to provide distribution maps of Cd and of nutrients (including Fe and S) at a sub-micrometer lateral resolution, and Cd speciation, respectively, particularly in the crease area. In addition to high-resolution maps, a grain dissection experiment was performed to provide quantitative information on Cd and nutrient concentrations in dissected grain tissues. The objectives of the study were to (i) precisely characterize the spatial distribution of Cd in durum wheat grain, (ii) investigate the differences between the distribution of mineral nutrients based on current knowledge of their loading, storage and functions in the grain, and (iii) examine if there are Cd-enriched tissues that could be removed by adequate milling processes without having too much impact on the nutritional value of the durum wheat flour.

Materials and methods

Grain production

Mature grains of durum wheat (Triticum durum cv. Sculptur) were harvested from hydroponically grown plants exposed to 100 nM Cd(NO 3 ) 2 . The composition of the nutrient solution and the hydroponic culture procedure are the same as those described in [START_REF] Yan | Contribution of remobilization to the loading of cadmium in durum wheat grains: impact of post-anthesis nitrogen supply[END_REF] (for details see supplementary comment S1). The average concentration of Cd in the grains was 2.5 ± 0.1 µg g -1 dry weight (DW). As a control for mapping, grains with a background level of Cd (0.0024 ± 0.0019 µg g -1 DW) were collected from plants grown under the same conditions but with no Cd added to the nutrient solution. Neither Cd toxicity nor mineral deficiency symptoms was observed in plants over the whole culture period. The spikes were freeze-dried at -60 ˚C for 72 hours before the grains were harvested. All the grains used in this experiment were taken from this pool, whose characteristics are summarized in supplementary Table S1.

Preparation of thin sections by cryomicrotomy

Grains were soaked for 5 to 6 hours at 4 °C. For LA-ICP-MS, grains were then embedded in O.C.T compound (tissue-teck) at low temperature (~ -30 °C) and 40 µmthick transversal (across root or leaf primordia of the embryo) as well as longitudinal (alongside the crease) sections were prepared by cryomicrotomy (Leica CM 1950).

The thin sections were directly deposited on glass slides and air dried. Before LA-ICP-MS analysis, the structural integrity of the grain section was checked under the microscope (Fig. 1-2, supplementary Fig. S1). For µXRF and µXANES, 16 µm-thick sections were cut at ~ -50 °C with a cryomicrotome (Leica LN22) cooled with liquid nitrogen. The frozen sections were sandwiched between two Ultralene ® films, and mounted directly on the sample holder immediately before measurement. They were kept frozen (-170 °C) during transfer to the analysis chamber and throughout the synchrotron measurements using a passively cooled liquid nitrogen cryostat.

LA-ICP-MS imaging

Elemental analysis of the grain section was achieved by coupling an NWR 213 laser ablation system equipped with a TV2 ablation cell (ESI, Freemont, CA) with a 7700 cs ICP-MS (Agilent, Tokyo, Japan) with Pt cones. A helium flow of 800 mL min -1 was used to transport the ablated material directly into the dry plasma of the ICP-MS. ICP-MS parameters (torch position, carrier gas flow rate and ion lense voltage) were tuned for each set of experiments by ablating the 612 NIST glass reference material while monitoring the 238 U and 232 Th signals. Detection limits of monitored isotopes are commonly in the range of 0.0001-0.1 µg g -1 [START_REF] Becker | Applications of inductively coupled plasma mass spectrometry and laser ablation inductively coupled plasma mass spectrometry in materials science[END_REF]. The grain sections were ablated using a Nd:YAG laser source at 213 nm with a 20 Hz repetition frequency, a 10 µm x 10 µm square laser beam, a fluency of 12 J cm -2 (20% of delivered energy), and a scan speed of 10 µm s -1 . Samples were entirely ablated in a line-by-line scan mode with a distance between lines of 20 µm (non-ablated lines). 114 Cd and other isotopes ( 31 P, 39 K, 55 Mn, 63 Cu, 64 Zn, 98 Mo) were measured with a sampling time of 1 s while maximizing the integration time for 114 Cd (0.59 s). A csv file reporting isotope intensities versus time (then converted to the position at the surface of the sample) was recorded for each ablated line. A specific program was designed and coded to merge the files and build the maps of the different isotopes. For each map, the relative intensity was calculated by dividing the raw signal by the maximum value.

Synchrotron µXRF imaging and µXANES analyses

Micro-XRF and Cd L III -edge µXANES were performed on the ID21 scanning X-ray microscope at the European Synchrotron Radiation Facility (ESRF, Grenoble, France).

The X-ray beam was monochromatized with a Si(111) two-crystal monochromator and focused using Kirkpatrick-Baez mirrors on the sample with a lateral resolution of 0.4 µm (V) x 1.4 µm (H). Measurements were made in a vacuum and in cryogenic conditions using a cryostat passively cooled with liquid nitrogen [START_REF] Cotte | The ID21 X-ray and infrared microscopy beamline at the ESRF: status and recent applications to artistic materials[END_REF].

To avoid potassium interference (K-edge at 3608 eV) with Cd L lines, chemical mapping was performed in two stages: Cd-S-P-Mg at 3570 eV and Fe-Mn-Ca-K at 7200 eV. In both cases, maps were obtained by scanning the samples with a 2 µm step (1 µm for the crease) and a counting time of 100 ms per point. The fluorescence signal was collected with a Silicon Drift Diode XRF detector (SGX Sensortech with an active area of 80 mm 2 ) and normalized by the incident photon intensity. Elemental mass fractions were calculated from fundamental parameters with the PyMca software package, applying pixel-by-pixel spectral deconvolution to hyperspectral maps normalized by the incoming flux (Sole et al. 2007). The incoming flux was monitored using a drilled photodiode previously calibrated to 3570 and 7200 eV. In PyMCA, the reference incoming flux was set at 5x10 10 photons s -1 , and XRF detector parameters were set to an active area of 0.7746 cm 2 and 4.65 cm sample to XRF detector distance.

Active area and sample to detector distance were calibrated using an AXO standard (http://www.axo-dresden.de/products/highprecision/reference.htm) that contains Fe and Pd as reference to the elements analysed in the wheat grains. The sample matrix was assumed to be cellulose/ice at a density of 1.5 g cm -3 . The sample thickness was set at 16 µm using a cryomicrotome. In order to filter out the pixels with poor signal to noise, each pixel was tested to have a signal in the detector element's region of interest ≥

. After filtering the poor signal quality pixels, the calculation of limit of detection (LOD) was performed for each pixel and the highest LOD calculated was used to set the color scale bars in the elemental maps.

The LOD maps and the elemental maps are shown in supplementary Fig. S4 andS5.

The LOD values obtained were (in mg g -1 ) 0.16 for Cd, 0.31 for S, 1.95 for P, 32 for Mg, 0.41 for Fe, 0.47 for Mn, 2.35 for Ca and 7.02 for K.

Cadmium L III -edge µXANES spectra of the areas of interest of the grain section were recorded in fluorescence mode using a SDD XRF detector (SGX Sensortech with an active area of 80 mm 2 ) in the range 3520 and 3590 eV with an energy step of 0.5 eV and a counting time of 100 ms. The normalized spectra were compared to Cdreference compounds we collected previously on the same beamline [START_REF] Isaure | Localization and chemical forms of cadmium in plant samples by combining analytical electron microscopy and X-ray spectromicroscopy[END_REF][START_REF] Isaure | Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the nonaccumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques[END_REF][START_REF] Penen | Pools of cadmium in Chlamydomonas reinhardtii revealed by chemical imaging and XAS spectroscopy[END_REF]) and simulated by linear combination fits (LCFs) of the Cd references (fingerprint approach). The quality of the fit was estimated by the normalized residual sum of squares (Nss) and the precision on the proportion of Cd species was estimated to be 10% as described in [START_REF] Isaure | The aquatic hyphomycete Heliscus lugdunensis protects its hyphae tip cells from cadmium: A micro X-ray fluorescence and X-ray absorption near edge structure spectroscopy study[END_REF].

Grain dissection and analysis of dissected grain fractions

Five fractions of wheat grain were considered: (i) the outer periphery (OP), which is loosely attached to the grain and mainly corresponds to the pericarp epidermis, hypodermis, and degenerated parenchyma [START_REF] Bechtel | Development, structure, and mechanical properties of the wheat grain[END_REF]), (ii) the inner periphery (IP) comprising the remaining layers that strongly adhere to the endosperm (cross and tube cells, seed coat, nucellar remnants and the aleurone layer), (iii) the germ (GM) including the scutellum and the embryo axis, (iv) the starchy endosperm (SE), and (v) the crease (CR) tissues comprising the periphery layers embedded in the crease of the grain. Because the IP parts stick tightly to the starchy endosperm, it was impossible to experimentally separate the whole IP from the whole SE while being sure that their respective mineral contents were not affected by cross-contamination.

Consequently, the concentrations of the element were determined on a subsample of the core of the starchy endosperm free from IP contamination. The quantity of a given element in the IP was estimated by calculating the difference between the quantity in the whole grain and the sum of the quantities in OP, GM, CR and SE. The concentration was then derived by dividing by the biomass of the IP (for details see supplementary comment S2).

The elemental composition of the fractions was determined as follows. A mixture of 0.8 mL of 30% H 2 O 2 and 0.2 mL of 67.5% HNO 3 was added to each sample fraction.

The mixture was left at room temperature overnight for preliminary digestion.

Digestion was accomplished by heating the mixture in an oven using a temperature gradient of 60 ˚C for 30 min, 80 ˚C for 45 min, and 100 ˚C for 90 min. After cooling and filtering through 0.2-μm polycarbonate membrane filters, the solutions were diluted to 10 mL with ultrapure water. The concentrations of Cd, Zn, Cu, Mn, Mo, and Fe in the digests were determined by ICP-MS (7700x, Agilent Technologies), and those of Ca, K, Mg, and P were determined by ICP-OES (ACTIVA, Horiba Jobin

Yvon) by the central analytical service of the University of the Basque Country. The results were validated using procedural blanks and the NIST Standard Reference Material 1573a. The recovery rates of reference values were higher than 90% for all elements.

Results

LA-ICP-MS elemental imaging in durum wheat grains

The maps of cadmium ( 114 Cd), zinc ( 64 Zn), copper ( 63 Cu), manganese ( 55 Mn), molybdenum ( 98 Mo), potassium ( 39 K), and phosphorus ( 31 P) distributions are shown for the cross sections at the level of the leaf (Fig. 1) or root (Fig. 2) primordia and for the longitudinal section alongside the crease (supplementary Fig. S1). The germ, crease, grain periphery, and starchy endosperm (SE) were identified on the different maps. Two layers were visible at the grain periphery: the outer (OP) and the inner (IP) periphery. In the germ, it was possible to distinguish the scutellum facing the endosperm and the embryo axis, including the coleoptile and coleorhiza surrounding the leaf and root primordia.

The starchy endosperm generally had the lowest intensities and the IP, the germ and the crease had the highest, regardless of the element. In detail, the OP had a detectable signal mainly for Cd, Mn and Zn. Except at the bottom of the grain close to the root primordia (Fig. 2), high abundances of all the elements, particularly K, P, Cd, Mn and Zn, were found in the IP layers. In the crease, which is visible only on the radial cross-sections (Fig. 1, 2), the signal intensities of Cd, Zn and Mn was very high but the exact location varied with the element and with their position along the embryo axis. On the cross section made at the level of the shoot primordia (Fig. 1), Cd was abundant in all the crease layers, Zn was more abundant in the innermost layers, presumably the modified aleurone layer, whereas Mn was more abundant in the outermost layers, presumably the nucellar projection, the pigment strand and the vascular bundle area. Hotspots of Cd, Mn and Cu were identified on the cross section at the level of the root primordia (Fig. 2) and attributed to the nucellar projection and/or the pigment strand. Zinc was highly abundant in the crease tissues on each side of the endosperm cavity, but the signal intensity of Zn was lower in the location assumed to be the pigment strand and/or in the vascular bundle area.

In the germ, the scutellum was particularly homogeneously enriched in P, Cu, K and to a lesser extent in Cd (Fig. 1-2, supplementary Fig. S1). In the scutellum, Zn was preferentially localized in the part facing the endosperm whereas Mn tended to preferentially accumulate on the opposite side, facing the shoot or root primordia, although to a lesser extent. In the embryo axis, the root cortex (and possibly the epidermis) showed stronger signals of Cd<Zn<Mn<Mo. Markedly high intensities of molybdenum were observed in the periphery of the root primordia (Fig. 2, supplementary Fig. S1). Finally, the starchy endosperm had always the lowest signals.

The lower abundances of elements in the SE than in the peripheral layers of the grain were clearly visible on the maps and in the signal intensity profile from the inside to the outside of the grain (supplementary Fig. S2). The difference between these and the other parts was (Zn, Mn, P, K) > (Cu, Mo) > Cd.

The accumulation of Cd in the SE was also visible when we compared the map of Cd in a contaminated grain with that in the uncontaminated control sample (supplementary Fig. S3). Figure 1 and supplementary Fig. S2 suggest a gradient in Cd intensity IP > outer endosperm neighboring IP > core of the starchy endosperm. It can be seen that the Cu signal in the endosperm was slightly higher around the crease (Fig. 1). Tentative grouping of the elements according to the most discriminating hotspots could be as follows: Cd, Mn and Zn accumulated particularly in the crease; P, Cu and K showed a stronger and more homogeneous accumulation in the scutellum; Mo was preferentially located in the periphery of the root primordia.

Synchrotron µXRF elemental imaging

Elemental maps of a transversal section with a zoom on the crease are shown in supplementary Fig. S4 and S5 and tricolor maps in Fig. 3. The highest concentration of Cd was clearly located in the crease, where its concentration locally reached 600 µg g -1 . The high resolution lateral map of the crease pinpointed a "curved zone" assumed to be the pigment strand [START_REF] Drea | Systematic spatial analysis of gene expression during wheat caryopsis development[END_REF] where Cd was particularly concentrated (red arrows in Fig. 3). Several hotspots where Cd and P co-localized were observed in the vascular bundle area (white arrows in Fig. 3). In contrast, no Cd was detected elsewhere in the grain, probably because the concentration of Cd was far lower in the other grain parts (endosperm, germ and peripheral tissues), as suggested by LA-ICP-MS maps. Sulfur was mainly located in the crease -in the vascular bundle area but also in the pigment strand and in the adaxial nucellar projection -as well as in the root primordia and in the endosperm, particularly in the outward endosperm, in line with results obtained by De Bier et al. (2016). High concentrations of P were found in the aleurone layer, where P-enriched vacuoles were visible, and in the scutellum. Iron was concentrated in the vacuoles of the aleurone cells (like P) and in the abaxial nucellar projection. The outward scutellum was also enriched in iron.

Manganese showed a different pattern with preferential localization in the vascular bundle area, in the outer periphery and, to a lesser extent, in the root primordia.

Calcium was distributed mainly as spots in the endosperm and in the outer periphery, while K was mainly located in the aleurone layer and in the germ (notably in the scutellum).

Cadmium speciation by Cd L III -edge µXANES

Cadmium µXANES was performed on the distinct areas of the crease and compared to Cd-references to probe the Cd ligands. Spectra collected on the various Cd+P hotspots were averaged and showed an edge with a peak characteristic of O ligands (supplementary Fig. S6). The global spectrum was satisfactorily reproduced by 62%

Cd-O and 27% Cd-cystein with O ligands provided by carboxyl ligands from organic acids. Although phosphate ligands produced poorer spectral agreement, it was difficult to unambiguously exclude these ligands. The averaged spectrum collected in Cd hotspots in the pigment strand was fitted with 43% Cd-O and 42% Cd-cystein while the averaged spectrum collected in the (Cd, S)-diluted areas of the vascular parenchyma was fitted with 27% Cd-O and 63% Cd-cystein. In contrast, no Cd absorption edge was detected in the nucellar projection, in the aleurone layer or in the endosperm because of their much lower concentration of Cd.

Concentrations and amounts of elements in the dissected grain parts

Figure 4 shows how the concentration of each element differed among the different grain parts. None of the elements studied were homogeneously distributed in the wheat grain. Three groups were distinguished among grain parts. The concentrations of SE were the lowest of all elements, clearly lower than the corresponding concentration of the whole grain (normalized concentration <1). The concentrations of the second group of grain tissues including the GM and IP were the highest of all the elements, 2.2 to 8.5 times higher than the concentration of the whole grain.

Concentrations in the third group, OP and CR, were between the two, either above or below the concentration of the whole grain, depending on the element. It is worth noting that the crease had by far the highest concentrations of Cd, Mn and Zn, with concentrations respectively 16.6-, 19.2-and 17.7-fold higher than the corresponding concentration of the whole grain. Concentrations obtained by µXRF and chemical analyses of dissected tissues differed but were not inconsistent since µXRF gives a local concentration in an area of a few µm 2 while chemical analyses provides information on a bulk tissue that can be highly heterogeneous. Generally, the ranking of grain tissues according to the concentrations of a given element was consistent with tendency for concentrations of Cd, Cu and Mo to be relatively the highest in the SE.

This characteristic may also be true for S, as suggested by µXRF maps (Fig. 3), but its concentration was not determined in dissected grain tissues in the present study.

Supplementary Fig. S7 shows how the amount of each element was partitioned among the different parts of the grain. SE+IP were the two main fractions that together contained between 52% (Mn) and 89% (Mg) of the total quantity of the grain. Hence, 69% of Cd in the grain was in SE+IP. Although IP represents only 10% of the grain dry weight, it accounted for between 22% (P) and 78% (Mg) of the total quantity of the element (30% of Cd). Conversely, SE, which is a main grain fraction based on biomass (83% of total, on average) accounted for only 25% (Ca) to 54% (Mo) of the total (39% of Cd). This reflects the high concentration of elements in the IP versus their very low concentration in the SE. The crease, which only accounted for 0.9% of grain DW, contained more than 15% of the Cd, Mn and Zn in the grain. The germ accounted for 3% of the grain biomass but contained more than a quarter of total P and 15-18% of total Cu, Mn, and Fe. The germ accumulated only 7% of total Cd. This is both the lowest contribution of the germ to the total quantity of an element and the lowest pool of Cd in the grain.

Discussion

Is nutrient accumulation in the crease evidence for their highly regulated loading into the seed?

In cereals, inorganic elements are mainly transported to the grain via the phloem [START_REF] Frick | Adaptive value of the xylem discontinuity in partitioning of photoassimilate to the grain[END_REF]. They are unloaded from the phloem all along the crease, transported through the apoplast and symplast of the chalaza and nucellar projection cells towards the endosperm cavity, a space filled with water and nutrients that are absorbed by the modified aleurone cells of the endosperm [START_REF] Wang | The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity[END_REF][START_REF] Patrick | Compartmentation of transport and transfer events in developing seeds[END_REF][START_REF] Borg | Iron transport, deposition and bioavailability in the wheat and barley grain[END_REF][START_REF] Yu | Structural development of wheat nutrient transfer tissues and their relationships with filial tissues development[END_REF]. In the early growth stage of the grain, it is possible that nutrients are also delivered to the endosperm from the whole pericarp through the seed coat by the dorsal and lateral bundles [START_REF] Fisher | Persistence of non-crease protophloem strands in the developing wheat grain[END_REF]).

However, this process is limited in time because the dorsal and lateral bundles degenerate and because the seed coat cuticulizes. The crease consequently becomes the main entry route for nutrients to the seed. In the crease region, part of the chalaza, namely the pigment strand, differentiates into an apoplastic barrier through the accumulation of hydrophobic substances and cell wall lignification. One function of the pigment strand may be to limit the backflow of nutrients (but not of water) from the seed to the maternal tissue. The seed is then isolated from the pericarp, seed coat and nucellar remnants [START_REF] Zee | Studies on the ontogeny of the pigment strand in the caryopsis of wheat[END_REF]. Nutrients can still be actively delivered to the seed via the symplastic route thanks to the transfer cells [START_REF] Wang | The cellular pathway of photosynthate transfer in the developing wheat grain. II. A structural analysis and histochemical studies of the pathway from the crease phloem to the endosperm cavity[END_REF] that enable control of the delivery.

Metal transporters coded by the genes of metal homeostasis are not completely specific and can transport Zn, Fe, Mn, Cd, Pb and Cu with variable affinity, explaining why the distribution of some metals in grains is similar, as is the case of Mn and Zn here. For example, HMA(1,2,3,10) pump Cd/Zn/Co/Pb across the membrane against their electrochemical gradient whereas HMA(4 to 9) operate on Cu/Ag [START_REF] Tauris | A roadmap for zinc trafficking in the developing barley grain based on laser capture microdissection and gene expression profiling[END_REF][START_REF] Bashir | Regulating subcellular metal homeostasis: the key to crop improvement[END_REF]. From the transversal sections of LA-ICP-MS maps obtained in our work, and more specifically at the shoot primordia level, it is possible to infer that Zn was more efficiently transferred from the phloem to the aleurone layer than Mn whose signal was higher in maternal tissues than in the aleurone layer. Based on current knowledge of putative metal-chelates in phloem [START_REF] Harris | A speciation model of essential trace metal ions in phloem[END_REF][START_REF] Khan | Moving toward a precise nutrition: preferential loading of seeds with essential nutrients over nonessential toxic elements[END_REF][START_REF] Flis | Inventory of metal complexes circulating in plant fluids: a reliable method based on HPLC coupled with dual elemental and high-resolution molecular mass spectrometric detection[END_REF], one possible explanation is that yellow strip-like transporters can transport the Zn-nicotianamine (Zn-NA) chelates from the phloem to the endosperm cavity and/or from the endosperm cavity to aleurone cells [START_REF] Palmgren | Zinc biofortification of cereals: problems and solutions[END_REF]) but no -or very little -Mn, which has a much lower affinity for NA than Zn (pK= 8.8 for Mn, pK= 17.4 for Zn; [START_REF] Benes | Metal complex formation by nicotianamine, a possible phytosiderophore[END_REF].

Relationship between the distribution and the function of inorganic nutrients in durum wheat grain

The endosperm is the reserve tissue of the seed but was seen to have the lowest concentrations of elements. This apparent paradox is however sound from a functional point of view. Indeed, at maturity, and therefore during germination, SE is a dead tissue and if inorganic nutrients were stored in SE, their concentrations would be very high so that transport by diffusion could cover the requirements resulting from the growth of the embryo. Given the size of the endosperm (80% of the seed DW), high concentrations also mean huge amounts of inorganic nutrients, which may not be available. High concentrations of inorganic elements in the endosperm may also affect starch synthesis and proper protein folding in which starch granules are embedded [START_REF] Mills | Chemical imaging: the distribution of ions and molecules in developing and mature wheat grain[END_REF]. Therefore, it is functionally more efficient to store inorganic nutrients in the aleurone layer [START_REF] Regvar | New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy[END_REF] because at germination, the aleurone cells are alive and can actively transfer nutrients to the germ [START_REF] Bechtel | Development, structure, and mechanical properties of the wheat grain[END_REF]. Aleurone cells (which are part of the IP fraction) have countless globoids of phytic acid combined with metal elements such as Zn, Mn, Cu, Fe, and Mg [START_REF] O'dell | Distribution of phytate and nutritionally important elements among the morphological components of cereal grains[END_REF][START_REF] Cakmak | Enrichment of cereal grains with zinc: Agronomic or genetic biofortification?[END_REF][START_REF] Ficco | Phytate and mineral elements concentration in a collection of Italian durum wheat cultivars[END_REF][START_REF] Brier | Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging[END_REF]) allowing the storage of both P and metals. The germ also contained high concentrations of elements, which can be interpreted as a starter pool for germination and for the initial growth of the seedling tissues. Although SE had the lowest concentrations of elements, likely reflecting the concentrations required for its metabolism, SE was on average the second pool of elements based on quantities, due to the important contribution of this tissue to the grain DW. Compared to the IP, SE is a significant pool of elements that can be mobilized more slowly, along with the mobilization of C and N reserves of the starch/protein matrix.

It is striking that the great majority of Cu accumulation took place in the scutellum and secondarily in the IP. This matches the distribution of P and is consistent with the high affinity of Cu for phytates, higher than that of Cd and Mn but equivalent to that of Zn [START_REF] Crea | Formation and stability of phytate complexes in solution[END_REF]. The ability of copper to change its redox state in biological conditions makes the control of its reactivity particularly important. Furthermore, in plants, excluding Cu sorbed onto the root apoplast, most Cu is contained in the plastocyanin of chloroplasts for photosynthesis, which is not among the plant's top priorities during early germination. From a functional point of view, it makes sense that Cu is stored in the scutellum and not in the embryo axis contrary to Zn and Mn, which are involved in many more enzymes during early germination, in particular those required for growth, protein synthesis, cell elongation, ion absorption and resistance to stress [START_REF] Broadley | Zinc in plants[END_REF][START_REF] Millaleo | Manganese as essential and toxic element for plants: transport, accumulation and resistance mechanisms[END_REF]. For example, Zn is required as a cofactor in over 300 enzymes [START_REF] Palmgren | Zinc biofortification of cereals: problems and solutions[END_REF]. Interestingly, Zn and Mn were highly concentrated in root primordia, which are the first organs to grow.

Molybdenum, which is required for both nitrogen assimilation and phytohormone synthesis [START_REF] Bittner | Molybdenum metabolism in plants and crosstalk to iron[END_REF], was also intensely located in the root cortex primordia.

Hence, the dissection data and the maps suggest particularly well integrated control of distribution among the grain tissues consistent with the fact that metals are essential for plant metabolism but also highly reactive and possibly toxic.

Cadmium distribution, loading and storage in durum wheat grain

To our knowledge, this is the first time that Cd has been mapped in wheat grain. In general, the distribution of Cd in the grain closely mirrored that of Zn and was characterized, notably, by very strong accumulation in the crease. The results of dissection showed that the concentration of Cd was on average 16-fold higher in the crease than in the whole grain, reaching 40 µg g -1 . The LA-ICP-MS and µXRF maps of the cross sections showed that Cd was not evenly distributed inside the crease and that distribution varied with the cutting axis. The µXRF distribution map obtained from a cut made at the level of root primordia showed that, on this axis, the vast majority of Cd accumulated in maternal tissues, notably in the pigment strand where it co-localized with S, and where its concentration locally reached 600 µg g -1 . This suggests that Cd loading in nucellar projection cells strongly limits its allocation to the seed, perhaps even more so than that of Mn. LA-ICP-MS maps showed that Cd was relatively more abundant in the endosperm than most other elements. The dissection results confirmed that the normalized concentration of Cd in the endosperm was higher than that of most nutrients, notably Fe and Zn, and showed that 40% of the total Cd in the grain was stored in the SE. This pattern may be related to the protein network of the SE that embeds starch granules including glutenins and gliadins [START_REF] Mills | Chemical imaging: the distribution of ions and molecules in developing and mature wheat grain[END_REF]. Indeed, Cd has a high affinity for S-containing compounds and, accordingly, the distribution pattern of Cd shown in Fig.

Speciation of

1 mirrors that of S in Fig. 3. In rice grains, Cd is associated with S-containing amino acids including cysteine and methionine [START_REF] Wei | Cadmium distribution and characteristics of cadmium-binding proteins in rice (Oryza sativa L.) kernel[END_REF]. The thiol groups of these amino acids are involved in the disulfide bonds that control protein folding and may be preferential sites of Cd binding and retention thereby explaining the relatively high concentration of Cd in the endosperm. Consistent with this hypothesis, the inward decreasing gradient of Cd in the endosperm may reflect the protein gradients in the SE, which are more concentrated in the subaleurone layer than in the inner parts [START_REF] Tosi | Distribution of gluten proteins in bread wheat (Triticum aestivum) grain[END_REF].

Development of new milling technologies to reduce the level of Cd in durum wheat products

New milling technologies for grain could improve the sanitary quality of durum products by removing the grain tissues that are rich in Cd. However, since there is no tissue in the grain of durum wheat that selectively accumulates Cd, a choice will need to be made between reducing the concentration of Cd and maintaining the nutritional quality of the grain. If the priority is to reduce the grain Cd concentration, for instance because the grain Cd concentration is high or because the grains are intended to be consumed by a population particularly exposed to Cd, the best option would be to remove all fractions except the starchy endosperm, which means producing "white semolina". According to the calculations made based on our dissection experiment (supplementary Table S2), this would reduce the concentration of Cd by more than 50% but would simultaneously cause huge nutrient losses, particularly of Mg, Fe, Mn and Zn. If the priority is to reduce the grain Cd concentration while minimizing the concomitant loss of inorganic nutrients, the best option would be to selectively remove the outer periphery and the crease, which means saving the SE+IP (with or without the germ) for the downstream transformation. This would reduce the Cd concentration by 24% while limiting the loss of P, K, Mg, Cu, Fe and Mo to less than 10%. Only the concentrations of Mn (-29%) and Zn (-21%) would be significantly reduced.

Conclusions

In this study, we combined two high-resolution imaging approaches (LA-ICP-MS and µXRF) with a dissection approach to describe the distribution of Cd and seven inorganic nutrients in durum wheat grain. The three approaches made it possible to identify high accumulation of Cd in the crease, and inside the crease, mainly in maternal tissues. Loading of Cd into the grain is therefore largely constrained, as is that of Fe and Zn. To control the accumulation of Cd in the grain without affecting that of Zn and Fe (or vice versa), the limiting stages of their loading into the grain and the associated physiological processes will have to be identified. The similarities observed between the µXRF maps of Cd and S distribution, particularly in the endosperm, as well as the results of µXANES showing that Cd is mainly linked to sulfur ligands in the vascular bundle area, suggest that sulfur fertilization could be a way to reduce the allocation of Cd to the grain as well as its accumulation in the endosperm in durum wheat. Molecular speciation measurements of Cd such as those conducted by [START_REF] Persson | Molecular speciation and tissue compartmentation of zinc in durum wheat grains with contrasting nutritional status[END_REF] will deepen the understanding of processes behind endosperm Cd accumulation. The preferential accumulation of Cd in certain grain tissues also opens the way for reducing the Cd content of durum wheat-derived products by adapting milling processes. However, because Cd accumulates in the same parts of the grain as several nutrients (including Zn) these adaptations will also alter the nutritional quality of the resulting flour.

  Cd by µXANES suggested various possible Cd ligands in the crease, probably corresponding to different forms of Cd transport or storage. It seems that Cd is nearly half bound by thiols in the Cd-enriched pigment strand while in (Cd, S)diluted areas of the vascular parenchyma this proportion increases to 63%. Assuming that the speciation of Cd in the vascular parenchyma mirrors that in the phloem vessels, this would mean that Cd is significantly associated with S ligands in the phloem of durum wheat, in agreement with the literature. Indeed, thiols were identified as Cd ligands in the phloem of the Cd hyperaccumulator Arabidopsis halleri and of the Cd non-accumulator Arabidopsis lyrata[START_REF] Isaure | Evidence of various mechanisms of Cd sequestration in the hyperaccumulator Arabidopsis halleri, the nonaccumulator Arabidopsis lyrata, and their progenies by combined synchrotron-based techniques[END_REF]. Thiol ligands were also found in the phloem of Brassicacea napus where Cd was assumed to be transported mainly as phytochelatin-Cd and glutathione-Cd complexes(Mendoza-Cozatl et al. 2008). Based on this result, one possible explanation for the strong accumulation of Cd in the maternal tissues of the crease is that transporters involved in metal loading in the nucellar projection and/or metal unloading in the endosperm cavity (such YSL and ZIP proteins) do not carry any (or only very few) metals bound to thiol-containing peptides such as Cd.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. LA-ICP-MS distribution maps of 114 Cd, 64 Zn , 63 Cu, 55 Mn, 98 Mo, 39 K and 31 P in mature durum wheat grains sectioned transversally at the position of the root primordia. The color scale represents different signal intensities, blue corresponds to the lowest relative intensity and yellow to the highest. The optical image (upper left)shows the different grain tissues visible in this transversal section: OP, outer periphery (likely the outer pericarp); SE, starchy endosperm; coz, coleorhiza; rp, root primordia; sl, scutellum; cr, crease.

Figure 3 .

 3 Figure 3. Synchrotron µXRF tricolor distribution maps of Cd-S-P, Cd-Mg-P, Fe-Mn-Ca and Fe-Mn-K in mature durum wheat grains sectioned transversally at the position of the root primordia. A more detailed map shows the distribution of Cd and P within the crease and the areas where µXANES spectra were collected (white arrows indicate typical positions of P and Cd hotspots, red arrows indicate typical positions of Cd hotspots, black arrows indicate typical Cd diluted areas). The optical image (upper left) shows the different grain tissues visible in this transversal section: OP, outer periphery (likely the outer pericarp); SE, starchy endosperm; al, aleurone layer; rp, root primordia; sl, scutellum. Within the crease (upper right), vb stands for vascular bundle area, ps for pigment strand and np for nucellar projection (either abaxial or adaxial). The maps of Cd, S, P and Mg were collected at an incident energy of 3570 eV, a step size of 2 µm (1 µm for the crease area) and a dwell time of 100 ms per point. The maps of Fe, Mn, Ca and K maps were collected at an incident energy of

Acknowledgements

This work was financially supported by the French National Research Agency through the CADON project (ANR-15-CE21-0001), by the CNRS-INSU EC2CO program through the CADMIGRAIN project, by ARVALIS-Institut du végétal through the CADUR project and by the French National Institute of Agronomic Research. Aquitaine Region and Feder are acknowledged for the funding of the laser ablation system through the AQUITRACE project (convention number 20131206001- 13010973). We also thank ESRF for the provision of beamtime and ID21 staff for their help in collecting synchrotron data. We are grateful to Juan Carlos Raposo from SGIker of UPV/EHU for technical and human support in ICP measurements, Cécile

Barron and Valérie Lullien-Pellerin for their advice on grain dissection protocols and 562 to the China Scholarship Council for the doctoral scholarship of Bo-Fang Yan. 563 7200 eV, a step size of 2 µm and a dwell time of 100 ms per point. Tricolor maps are displayed as quantitative maps in mg g -1 .

Figure 4. Concentrations of P, Ca, K, Mg, Cd, Cu, Fe, Mn, Mo, and Zn measured in the following dissected grain tissues: germ (GM), outer periphery (OP), inner periphery (IP), starchy endosperm (SE) and crease (CR). The concentration of a given element in a tissue was normalized by dividing the concentration of the tissue by that of the whole grain.