Brassica napus, Nasturtium officinale, Camelina sativa, Lepidium sativum, and Brassica oleracea seeds were purchased at a local market. B. napus and N. officinale plants were grown on soil in a greenhouse at 23°C under long-day conditions (16-h/8-h day/night cycle).

Iron staining was performed according to Roschzttardtz et al. (2009). Brassica napus, Nasturtium officinale, Camelina sativa, Brassica oleracea, and Lepidium sativum dry seeds or seeds from B. Napus and N. officinale at different developmental stage were vacuum infiltrated with fixation solution (2% w/v paraformaldehyde in 1 mM phosphate buffer pH 7.0) for 45 min and incubated for 16 h in the same solution. The fixated seeds were dehydrated with ascendant concentration of ethanol (50, 70, 80, 90, 95, and 100%), later were incubated 12 h with a solution of butanol/ethanol 1:1 (v/v), and finally were incubated 12 h with 100% butanol. Then, the seeds were embedded in the Technovit 7100 resin (Kulzer) according to the manufacturer’s instructions and thin sections (3 μm) were cut and were deposited on glass slides. For Perls/DAB staining, slides were incubated with 2% (v/v) HCl and 2% (w/v) K-ferrocyanide (Sigma–Aldrich) for 45 min For the DAB intensification, each glass slide was washed with distilled water, later were incubated in a methanol solution containing 0.01 M NaN₃ (Sigma–Aldrich) and 0.3% (v/v) H₂O₂ (Merck) for 1 h, and then washed with 0.1 M phosphate buffer (pH 7.4). For the intensification reaction, the glass slides were incubated between 10 and 30 min in a 0.1 M phosphate buffer (pH 7.4) solution containing 0.025% (v/v) H₂O₂, and 0.005% (w/v) CoCl₂^∗6H₂O (intensification solution). To stop the reaction each slide was rinsed with distilled water.

A microwave-assisted acid digestion was performed using 6.0 mL of concentrated HNO₃ (Winkler) and 1.0 mL of 30% (v/v) H₂O₂ added over 10–20 mg of seeds of each genotype. The colorless digestate were filled up to 15 mL using deionized water. The iron content of each genotype was measured in triplicate using inductively coupled plasma mass spectrometry (ICP-MS).

Thin sections were first stained with DAPI in order to visualize nuclei (Roschzttardtz et al., 2013), the same section was stained by Perls/DAB, as described above. Then were stained with toluidine blue. Each slide was incubated in a 0.5% (w/v) solution of toluidine blue for 2 min and rinsed with distilled water. The samples were observed and photographed with a Nikon Eclipse 80i microscope.

To evaluate iron concentration and distribution in dry seeds from closely related Arabidopsis species, first we determine iron content in dry seeds from Arabidopsis thaliana, Brassica napus and Nasturtium officinale (Figure 1A). Dry seeds were isolated, and a microwave-assisted acid digestion was performed. Figure 1B show iron concentration of each species determined by inductively coupled plasma mass spectrometry (ICP-MS). Iron concentration in Arabidopsis thaliana and Brassica napus dry seeds (70.4 and 64.6 μg of iron/g of seeds, respectively) was lower compared to Nasturtium officinale (116.6 μg of iron/g of seeds), suggesting that there is no relation between iron concentration and seed size, as Brassica napus have the biggest seeds (Figure 1A) but also have a similar iron content as Arabidopsis thaliana.

In order to determine where iron localizes in dry seed embryos from species closely related to Arabidopsis thaliana, we analyzed seven different plant species: Brassica napus, Nasturtium officinale, Camelina sativa, Arabidopsis thaliana, Lepidium sativum, Brassica oleracea and Brassica oleracea var. capitate (Figure 2 and Supplementary Figure S2). Embryos from dry seeds were isolated and fixed. Thin sections of different embryo regions were analyzed, in particular, cotyledon and hypocotyl. Figure 2 and Supplementary Figure S2 show Perls/DAB staining revealing iron distribution in embryos from these species. Cotyledons accumulate iron in vacuoles of cells surrounding provasculature (Figures 2A–D and Supplementary Figures S2A,B) as has been described before for Arabidopsis thaliana (Roschzttardtz et al., 2009). Provasculature complexity in cotyledon is clearly higher than Arabidopsis (Roschzttardtz et al., 2014, 2017). Iron accumulation was not observed in other cell types like protodermis and cortex cells in dry seed embryos (Figures 2A–D and Supplementary Figures S2A,B).

Interestingly, transversal sections of hypocotyl show differences in the number of cell layer where iron accumulates compared to Arabidopsis hypocotyl, where only one cell layer accumulates iron (Roschzttardtz et al., 2009). We found that at least two-cell layers accumulate iron in the hypocotyl of Brassica napus, Nasturtium officinale, Camelina sativa, Lepidium sativum and Brassica oleracea dry seed embryos (Figures 2E–G and Supplementary Figure S2C,D, respectively). Despite the differences in the number of cell layers where iron accumulates in dry seeds of Brassica napus, Nasturtium officinale, Camelina sativa, Lepidium sativum and Brassica oleracea compared to Arabidopsis thaliana, at subcellular level iron is stored in the vacuoles in cells surrounding provasculature (Figures 2A–D and Supplementary Figures S2A,B).

Analyses of embryonic root tips indicate that iron accumulates in different cells identified as endodermis-cortex cells (Supplementary Figure S3).

Supplementary Figure S1 shows the phylogenetic tree of the 5 species of Brassicaceae family used in this study. Interestingly, iron localization in vacuoles is a conserved trait in the analyzed plant species. As indicated above, differences were only observed in the number of cell layers that accumulate iron in the hypocotyls.

As we showed, iron accumulates in vacuoles in embryos of seven species of Brassicaceae (Figure 2 and Supplementary Figure S2). We used Brassica napus plants as model to study where iron localizes during seed development. B. napus produces bigger seeds compared with Arabidopsis plants allowing to study with more details different seed regions during its development (Figure 1A). In a phylogenetic point of view, B. napus is the more distant specie to A. thaliana used in this study. The fact that iron localizes in vacuoles of mature B. napus seeds is a strong indication that it is a conserved trait. Using A. thaliana as plant model it has been described that iron accumulates in embryo during seed maturation stages (Roschzttardtz et al., 2009), for this reason we analyzed three different stages of seed maturation, torpedo, bent cotyledon and mature stage before seed desiccation. In order to describe seed structures where iron accumulates before to be loaded into the embryo, we used in our analysis whole seeds including seed coat, endosperm and embryo. Whole seeds of B. napus containing embryos in different maturation stages as indicated above, were fixed and embedded in Technovit resin according to Section “Material and Methods.”

The analysis of seeds with torpedo embryo stages revealed that iron localizes mainly in nuclei of integument cells, free cell endosperm and in different cell layers in the embryo (Figure 3). At this embryo stage, Perls/DAB and Toluidine blue staining show that in protodermal, cortex, endodermis and provasculature cells iron is localized in nuclei (Figures 3A,B). As vacuoles look empty after both staining, our results suggest that vacuoles of embryo cells are not an important compartment for iron storage at this developmental stage. Interestingly, strong iron staining was observed in nuclei (one to three iron pools were observed) of the free cell endosperm surrounding the embryo (Figures 3C,D).

Later, in seeds containing bent cotyledon embryo stage, integuments no longer contain iron inside the nuclei (Figures 4A,B). In the embryo, the number of cells containing iron in nuclei decreases. In embryonic root tips and cotyledons, iron is localized in structures surrounding the nuclei (Figures 4A,B,E,F), while vacuoles are starting to load iron in the hypocotyl endodermis cell layer (Figures 4C,D).

In mature seeds before desiccation, iron does not localize in nuclei (Figure 5) and vacuoles are the principal compartments for iron storage (Figures 5B,D). At this late stage of seed development, cells adjacent to provasculature load iron and protodermis and cortex cells have not detectable iron as the previous seed developmental stages (Figure 5).

In order to determine whether iron is accumulated in nuclei before torpedo stages, we use N. officinale developing embryos. Our results indicate that in the heart stage of embryo development iron is not detectable by Perls/DAB staining in embryo cells. This result strongly supports the conclusion that iron localizes in nuclei of embryo cells in the torpedo stage (Supplementary Figure S5). Similar stages of seed maturation were analyzed using N. officinale. No differences in iron distribution were observed compared to B. napus (Supplementary Figure S4).

In order to confirm the subcellular compartments accumulating iron during development we performed consecutively staining with DAPI, Perls/DAB and toluidine blue on the same sections of different developmental stages of B. napus embryos. Iron accumulates in nuclei of different cell types in early maturation stages of seed development (Figures 6A–E). In bent cotyledon stage, iron is not longer detected in nuclei and accumulates in cytoplasmic structures (Figures 6K–O). Finally in mature stages, vacuoles of cells surrounding provasculature start to accumulate iron (Figures 6P–T). Details of unique cells are shown in Figure 6, indicating unambiguously were iron localizes in different cases, nuclei, cytoplasmic structures or vacuoles.