**5. Materials and Methods**

### *5.1. Plant Material and Total Element Concentration in Grain*

The grain of 20 di fferent wheat (*Triticum aestivum* L.) cultivars were obtained from the Agricultural Institute of Slovenia, with their agronomic characteristics reported in Table S1. Among these wheat cultivars, nine were awned (cv. Euclide, cv. Lukullus, cv. Vulkan, cv. Isengrain, cv. Renan, cv. Soissons, cv. Ingenio, cv. Bologna, and cv. Element) and 11 were awnletted (cv. BC Nina, cv. BC Renata, cv. Rosario, cv. Felix, cv. Renata, cv. Katarina, cv. BC Zdenka, cv. Anđelka, cv. Gracia, cv. Bastide, and cv. Super Zitarka). The plants were grown in the field of the Infrastructure centre Jablje (central Slovenia: 46◦8-59" N, 14◦33-31" E, 307 m above sea level) in 2014/2015 on pseudogley-gley soil type, which has a silty clay texture. The previous crop on the field site was grain maize (*Zea mays* L.). The field was fertilised with 205 kg·ha−<sup>1</sup> N (in 5 rations), 90 kg·ha−<sup>1</sup> P2O5 and 120 kg·ha−<sup>1</sup> K2O; 400–750 germinative seeds m<sup>−</sup><sup>2</sup> were sowed respecting interrow spacing of 12.5 cm. Trial layout was a randomized block design with four repetitions, with each plot having 7.5 m2. At maturity, the plants were harvested, the 1000-grain weight was determined (g), the grain was homogenised and milled or stored for the localisation analyses. The ground material was pressed into pellets (6–12 for each cultivar) using a pellet die and hydraulic press. Total concentrations of P, S, K, Ca, Mn, Fe, and Zn were measured in whole grain samples and in standard reference material NIST SRM 1573a (tomato leaves) for quality assurance using X-ray fluorescence, as described previously [48]. Based on the total Fe concentration in the whole grain of the 20 wheat cultivars, four contrasting cultivars were selected. Cultivars Katarina and Super Zitarka, the two awnletted wheat cultivars, had a larger total Fe concentration than the two awned cultivars Vulkan and Soissons.

### *5.2. Sample Preparation and Micro-XRF Mapping and Micro-XANES Analysis*

The grain of the four wheat cultivars was soaked in MiliQ water for 2 h at 4 ◦C. Whole grain was hand-cut transversely at the embryo location under a stereomicroscope into approximately 200 μm thick sections using new stainless-steel platinum-coated razor blades and frozen immediately in liquid isopentane (Sigma-Aldrich, St. Louis, MO, USA) cooled by liquid nitrogen [49]. Frozen-hydrated sections were sandwiched between two Ultralene ® foils (each 4 μm thick), deposited on custom made Cu holders and analysed at the ID21 beamline (ESRF, Grenoble, France) as described previously [33]. In short, measurements of Fe K-edge micro-XANES (recorded in the energy region from 7040 to 7250 eV) were performed on high Fe pixels (*n* = 2–9) identified by fast mapping of grain sections using synchrotron radiation micro-X-ray fluorescence (micro-XRF) at the same beamline. This approach enabled the best quality micro-XANES spectra of the two regions of interest in the wheat grain: the crease and the scutellum. Specific attention was paid to avoid radiation damage and photoreduction of Fe3<sup>+</sup> [50], as described in detail in [25,33].

The qualitative distribution maps of Fe, P, and S were quantitatively analysed, as described previously [51,52] and the quantitative distribution maps and co-localisation maps were generated with PyMCA software [53]. The Fe K-edge micro-XANES spectra were analysed with the IFEFFIT program package Athena [54], where linear combination fitting was performed using the following reference Fe2<sup>+</sup> and Fe3<sup>+</sup> complexes: Fe2<sup>+</sup> phytate, Fe2<sup>+</sup> nicotianamine, Fe2<sup>+</sup> sulphate, Fe3<sup>+</sup> phytate, Fe3<sup>+</sup> nicotianamine, Fe3<sup>+</sup> citrate and α-Fe3<sup>+</sup>OOH - oxide/hydroxide (goethite). The preparation and analysis of these reference Fe complexes has been described elsewhere [25,33], except for Fe nicotianamine standards, which were synthesized by mixing water solution of nicotianamine (CAS 34441-14-0, Santa Cruz Biotechnology, Inc., USA) with FeCl2 × 4H2O (Sigma-Aldrich) or FeCl3 × 6H2O (Sigma-Aldrich) water solution at the molar ratio of 10:1 in water. Final pH was adjusted to 6.5–7.0. XANES spectra of the reference compounds Fe2<sup>+</sup> nicotianamine and Fe3<sup>+</sup> nicotianamine were measured at XAFS beamline of synchrotron Elettra (Trieste, Italy) in transmission detection mode, on homogeneous pellets with optical thickness of about 2 above Fe absorption K-edge. Iron species and Fe ligands for each wheat cultivar were obtained by averaging across all tissue-specific XANES results in each cultivar.
