**2. Results**

### *2.1. Total Concentrations of Mineral Elements in Whole Wheat Grain*

A significantly larger total concentration of Ca and Mn, but significantly smaller total concentration of Fe was found in whole grain of awned wheat cultivars than in awnletted cultivars (Figure 1).

**Figure 1.** Variability in the total concentration of phosphorus (P), sulphur (S), potassium (K), calcium (Ca), manganese (Mn), iron (Fe), and zinc (Zn) in whole grain of wheat (*Triticum aestivum* L.) cultivars di ffering in the awn type (awned cultivars have long awns and awnletted cultivars have short or no awns) grown in the same field. Shown are boxplots representing 25th and 75th percentile of the data, with the middle line representing the median, whiskers representing the 5th and 95th percentile and the black dots representing outliers (*n* = 87 and *n* = 100 data points of nine awned and 11 awnletted wheat cultivars, respectively). Asterisks indicate significant di fferences between the awned and the awnletted cultivars (Student *t*-test; \*\*\* *p* < 0.001 and \* *p* < 0.05).

The total concentration of Fe in the awned wheat cultivars ranged from 50.8 to 94 mg·kg−<sup>1</sup> dry weight (1.85-fold variability) and in the awnletted wheat genotypes from 67.3 to 103 mg·kg−<sup>1</sup> dry weight (1.53-fold variability). Considering all wheat cultivars studied, total Fe concentration in whole grain varied 2.03-fold.

There was no di fference in 1000-grain weight (Table S1) and in concentrations of P, sulphur (S), potassium (K) and Zn in the whole grain between the awned and the awnletted wheat cultivar group (Figure 1), nor was there any apparent separation of awned and awnletted cultivars when the whole elemental profile was considered (Figure S1). The hierarchical clustering indicated that Fe and Zn were grouped apart from the rest of the mineral elements, among which P, K, and S grouped apart from Ca and Mn (Figure S1). A significant positive correlation was observed between grain concentrations of P and those of K, S, and Fe, but no significant correlation between grain concentrations of P and Ca, Mn, and Zn (Figure S2a). Positive correlation was found between grain concentration of Fe and Mn, and of Fe and Zn (Figure S2b).

Four wheat cultivars with contrasting Fe concentrations were selected for further in-depth analyses: Two awned cultivars (cv. Vulkan and cv. Soissons) of low-Fe accumulation (the average total concentrations in the grain was 73.4 and 77.0 mg Fe·kg−<sup>1</sup> dry weight, respectively), and two awnletted cultivars (cv. Katarina and cv. Super Zitarka) of high-Fe accumulation (the average total Fe concentrations in the grain was 83.7 and 91.3 mg Fe·kg−<sup>1</sup> dry weight, respectively; Figure 2). In agreemen<sup>t</sup> with observations for all wheat cultivars studied, there was a positive correlation between grain P concentrations and grain S, K, and Fe concentration (Figure S3a) and between grain Fe and grain Zn (but not Mn) concentrations (Figure S3b) in these four wheat cultivars.

**Figure 2.** Total iron (Fe), sulphur (S) and phosphorus (P) concentrations in the grain of wheat (*Triticum aestivum* L.) cultivars differing in the awn type (awned cultivars have long awns and awnletted cultivars have short or no awns) grown in the same field. Circles represent awned cultivars and triangles represent awnletted cultivars. The four cultivars (Katarina, Super Zitarka, Vulkan and Soissons) selected for further in-depth analyses are highlighted in colour. Shown are averages (*n* = 6–12). d.w.—dry weight.

### *2.2. Tissue-Specific Iron, Phosphorus and Sulphur Concentrations, Iron Speciation and Iron Ligands*

Iron species and Fe ligands were studied in two different regions of interests (Figure 3) of the frozen-hydrated grain cross-sections of the four wheat cultivars. The first region of interest comprised nucellar projection, modified aleurone, endosperm, transfer cells, and scutellum. The second region of interest comprised aleurone, scutellum, embryo, endosperm, and pericarp.

**Figure 3.** A representative wheat (*Triticum aestivum* L.) grain cross section with the two regions of interest (ROI) highlighted with red squares, namely ROI1 (the crease) and ROI2 (the scutellum). E—endosperm.

To identify higher Fe signal pixels—selected for subsequent micro-XANES analysis—the regions of interest were first subjected to a fast (to avoid photoreduction of Fe by the focused X-ray beam) micro-XRF mapping at the ID21 beamline at ESRF to localise Fe, P, and S (the quantitative maps are shown in Figures S4 and S5). By identifying Fe hotspots, the best signal-to-noise ratio in Fe K-edge micro-XANES spectra was ensured. In endosperm, the concentrations of Fe were too small (on average 3.5 mg·kg−<sup>1</sup> fresh weight in imbibed grains of awned cultivars and 11.4 mg·kg−<sup>1</sup> fresh weight in imbibed grains of awnletted cultivars) to yield micro-XANES spectra of su fficient quality. Similarly, larger Fe concentrations were found in aleurone and in pericarp of the awnletted cultivars (82.3 and 24.6 mg·kg−<sup>1</sup> Fe fresh weight, respectively) than in aleurone of the awned cultivars (49.2 and 12.2 mg·kg−<sup>1</sup> Fe fresh weight, respectively). By contrast, in embryo and nucellar projection the average Fe concentration of awned cultivars (33.8 and 160 mg·kg−<sup>1</sup> Fe fresh weight, respectively) was larger than in awnletted cultivars (12.5 and 92 mg·kg−<sup>1</sup> Fe fresh weight, respectively). In scutellum, both cultivars contained similar Fe concentration (60.5 mg·kg−<sup>1</sup> Fe fresh weight in awned cultivars and 64 mg·kg−<sup>1</sup> Fe fresh weight in awnletted cultivars).

The micro-XANES spectra from selected Fe hotspots (2 to 4 per section as indicated on the Fe, P and S co-localisation maps in Figures 4 and 5) were compared to the spectra of the Fe reference compounds and complexes (Figure S6; reported previously [25,33]).

**Figure 4.** Co-localisation images of iron (Fe) in red, sulphur (S) in green and phosphorus (P) in blue in the two regions of interest (ROI): the crease (ROI1) and the scutellum (ROI2) in the frozen-hydrated grain of wheat (*Triticum aestivum* L.) cultivar Vulkan (**a**) and Soissons (**b**), the awned wheat cultivars. × indicates pixels where Fe K-edge micro-XANES spectra were recorded and ¤ indicates where the selected Fe K-edge micro-XANES spectra (solid line) was recorded and is displayed on the right-hand side. The best linear combination fit (red dashed line) was obtained by the spectra of the reference Fe compounds. The relative amount of each component is given in parentheses. eV—electron volts; NA—nicotianamine. Scale bars = 200 μm. Quantitative distribution maps of Fe, P and S can be found in Figure S4.

**Figure 5.** Co-localisation images of iron (Fe) in red, sulphur (S) in green and phosphorus (P) in blue in the two regions of interest (ROI): the crease (ROI1) and the scutellum (ROI2) in the frozen-hydrated grain of wheat (*Triticum aestivum* L.) cultivar Katarina (**a**) and Super Zitarka (**b**), the awnletted wheat cultivars. × indicates pixels where Fe K-edge micro-XANES spectra were recorded and ¤ indicates where the selected Fe K-edge micro-XANES spectra (solid line) was recorded and is displayed on the right-hand side. The best linear combination fit (red dashed line) was obtained by the spectra of the reference Fe compounds. The relative amount of each component is given in parentheses. eV—electron volts; NA—nicotianamine. Scale bars = 200 μm. Quantitative distribution maps of Fe, P and S can be found in Figure S5.

The Fe K-edge micro-XANES spectra could be described as linear combinations of the Fe K-edge XANES spectra of the following Fe complexes: Fe2<sup>+</sup> phytate, Fe2<sup>+</sup> sulphate, Fe2<sup>+</sup> nicotianamine, Fe3<sup>+</sup> phytate, Fe3<sup>+</sup> nicotianamine, Fe3<sup>+</sup> citrate, and α-Fe3<sup>+</sup>OOH (Fe oxide-hydroxide; goethite). Relative amount of each Fe complex in the combination (Table S2) was obtained from the best fit with a ± 1% error for the Fe<sup>2</sup>+/Fe3<sup>+</sup> complex ratio and a ± 5% error for the Fe3<sup>+</sup> phytate/Fe3<sup>+</sup> non-phytate ratio.

On average, the four cultivars did not differ in the Fe species and Fe ligand composition (Figure 6a,c). Ferric species was predominant in all four cultivars, with 64% of the total Fe found in this form (Figure 6a), which was a cumulation of 26% being phytate ligand and 38% non-phytate ligands. The remaining Fe was present as ferrous species (36%) in all four cultivars, which was a cumulation of 27% bound to phytate and 9% to non-phytate ligands. In total, 53% of Fe was found bound to phytate and the remaining 47% to non-phytate ligands (Figure 6b and Table S2).

**Figure 6.** Average relative amounts (%) of iron (Fe) species, Fe ligands and Fe, phosphorus (P) and sulphur (S) concentration in the grain of wheat (*Triticum aestivum* L.) cultivars Vulkan and Soissons (awned wheat cultivars) and Katarina and Super Zitarka (awnletted wheat cultivars) (**<sup>a</sup>**,**c**,**<sup>e</sup>**) and in grain tissues (**b**,**d**,**f**). Phosphorus, Fe and S concentrations are in mg·kg−<sup>1</sup> dry weight (**e**) or fresh weight (**f**). In (**b**,**d**,**f**) results indicate average across all four cultivars.

A significant tissue-specificity for Fe speciation and Fe ligand composition was observed (Figure 6b,d). In all tissues studied, the majority of Fe species were ferric—except in nucellar projection, with equal contribution of ferric and ferrous species (Figure 6b). In the order of decreasing content of ferric species, the list of tissues is: pericarp < aleurone = scutellum < embryo < nucellar projection (Figure 6b). By the proportion of phytate ligands, the tissues were ordered as: aleurone < scutellum << embryo << nucellar projection = pericarp, with the latter two tissues having no phytate ligands, but only non-phytate ligands (Figure 6d and Table S2). Of non-phytate ligands in nucellar projection Fe3<sup>+</sup> citrate was most prominent (34%), followed by Fe2<sup>+</sup> nicotianamine (29%), Fe2<sup>+</sup> sulphate (22%) and Fe3<sup>+</sup> nicotianamine. By contrast, the pericarp contained mostly Fe3<sup>+</sup> oxide-hydroxide (52%), followed by Fe3<sup>+</sup> nicotianamine (23%), Fe2<sup>+</sup> sulphate (14%) and Fe3<sup>+</sup> citrate (12%). Of non-phytate ligands in other tissues, only Fe3<sup>+</sup> citrate was found, with the largest proportions in embryo (45%), followed by scutellum (30%) and aleurone (27%). No clear correlation between the total and local Fe, P and S concentration and the Fe ligand profile in tissues could be discerned (Figure 6e,f). The nucellar projection contained the largest concentration of Fe, while pericarp contained the smallest concentration of Fe, P and S (Figure 6f).
