Increased iron content in seeds is an important agronomic trait. This is due to the relevance of this element in seed production (Marschner, 2005; Roschzttardtz et al., 2011), embryo development, and seedling germination and growth (Lanquar et al., 2005; Kim et al., 2006), as well as in human nutrition (Murgia et al., 2012). In spite of this essential role, the prevalent low iron bioavailability in the soil of most of the main agricultural areas of the world, limits plant productivity, fertility, and even germination rates (Guerinot and Yi, 1994). Consequently, a substantial effort has been dedicated to unraveling the molecular bases controlling iron homeostasis, how plants incorporate and distribute iron throughout their organs, and how iron is stored in the seeds (Walker and Waters, 2011; Kobayashi and Nishizawa, 2012; Grillet et al., 2014; Flis et al., 2016; Tsai and Schmidt, 2017).

Iron distribution in seeds has been studied in monocots and Eudicotyledoneae. Using rice and wheat as models it was concluded that most of the iron in monocot plants is stored in the aleurone layer (Iwai et al., 2012; De Brier et al., 2016). In Eudicotyledoneae, the majority of work has been carried out in Arabidopsis. It has been estimated that approximately 50% of the seed iron content is stored in endodermal cells (Roschzttardtz et al., 2009; Schnell Ramos et al., 2013). Results from experiments using multiple iron-imaging methods showed that the vast majority of the seed embryo iron is located in vacuoles (Kim et al., 2006; Roschzttardtz et al., 2009; Schnell Ramos et al., 2013). This is in contrast to other plant tissues in which iron has also been detected in plastids, associated with ferritin, and in nucleoli (Roschzttardtz et al., 2011, 2013).

Iron accumulates steadily in the endodermal vacuole during the maturation stage of embryo development, and is subsequently used during post-germinative growth (Lanquar et al., 2005; Roschzttardtz et al., 2009). The defects shown by the vit1 and nramp3 nramp4 mutants under iron deficiency conditions suggest that proper iron storage seems essential for seed germination and post-germinative growth (Lanquar et al., 2005; Kim et al., 2006; Mary et al., 2015). These mutant plants are defective in iron loading into the vacuoles of endodermal cells during embryo maturation, a process mediated by the transporter AtVIT1 (VACUOLAR IRON TRANSPORTER1) (Kim et al., 2006); or in iron recovery from vacuoles in these cells via the AtNRAMP3 and AtNRAMP4 (NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN3 and 4) transporters during germination (Lanquar et al., 2005).

Closely related Brassicaceae embryos accumulate iron very similarly to Arabidopsis, i.e., in vacuoles of cells surrounding the embryo provasculature (Ibeas et al., 2017). However, reports on another Eudicotyledoneae, the legume Phaseolus vulgaris, showed broader iron localization. While iron hotspots were detected in cotyledons in a pattern resembling the provasculature, the metal was also evenly distributed in the rest of the organ (Cvitanich et al., 2010). This alternative way of accumulating iron in the seed could indicate that the storage of this nutrient in Eudicotyledoneae is more diverse than what has been reported to date. Considering the importance of Eudicotyledoneae as crops, and the need to improve the iron content in the edible parts (DeFries et al., 2015), we set to study iron distribution in embryos from a wide range of orders belonging to dicotyledonous plants. Our results show that the iron distribution pattern in the Arabidopsis embryo is not extended to all Eudicotyledoneae, it rather seems to be a derived character only observed in the Brassicaceae family. We also found that Caryophyllales has a distinct iron distribution pattern and that iron loading during Chenopodium quinoa seed development is different than what we had previously observed in Brassicaceae (Ibeas et al., 2017), suggesting there might be different strategies for the storage of iron in these seeds.

Dietary iron deficiency is a major issue for human health, affecting more than 2 billion people around the world (Murgia et al., 2012). Seeds are a pivotal source of iron for humans and animals (Roschzttardtz et al., 2017). Therefore, understanding the molecular mechanisms involved in iron loading and distribution in seeds is critical for the development of biotechnological approaches to improve seed iron content. Generation of biofortified crops could be a financially sound strategy to deliver nutrients to the population, as an alternative to the continuous governmental investment in fortification and supplementation programs (DeFries et al., 2015). Thus, genetic biofortification of staple crops could result in an environmentally friendly and cost-effective strategy for the improvement of nutritional health (Guerinot and Yi, 1994).

Pioneer studies, using SμXRF and a histochemical method for iron detection in plant tissues (Perls/DAB stain), showed that iron accumulates in the vacuoles of the endodermis cell layer of A. thaliana mature embryos (Kim et al., 2006; Roschzttardtz et al., 2009; Eroglu et al., 2017; Ibeas et al., 2017). While this distribution pattern is conserved in the rest of the Brassicaceae family (Ibeas et al., 2017), it is not present in legumes (Cvitanich et al., 2010). These observations suggest that embryos may have multiple ways to store iron. Consequently, by exploring this diversity, we should be able to propose suitable models to dissect the molecular mechanisms of seed iron storage and eventually translate these findings to crops of commercial and nutritional value. Toward this end, we used Perls/DAB staining to determine iron distribution in several plant species along the Eudicotyledoneae phylogeny. As a result, we obtained the first phylogenetic study of iron distribution in plant seed embryos. The results indicate that seed embryos belonging to different Eudicotyledoneae orders accumulate iron in several cell layers, with more diversity that what can be inferred by simply using Arabidopsis as model. In particular, cortex cells accumulate iron in all the embryo species analyzed except for Brassicales families Capparaceae, Cleomaceae and Brassicaceae. These results strongly suggest that iron distribution in cortex cells in embryos from Eudicotyledoneae embryo plants is a conserved characteristic from a phylogenetic point of view. We used S-μXRF as a second and independent method to confirm that in quinoa embryos, iron accumulates in several cell layers including the cortex and endodermis cells (Figure 3B).

Quinoa seed has high nutritional value and has recently started to be used as a novel functional food (Abugoch, 2009). This plant contains 15 mg of iron per 100 g of seeds, covering the daily iron needs of infants and adults (National Academy Press, 2001; Konishi et al., 2004). We evaluated iron accumulation during C. quinoa seed development and found that it does not accumulate iron in any seed tissue during the early developmental stages (Figure 4). This is in contrast with what was previously described for B. napus seeds, in which strong iron staining was observed in the nuclei of the free cell endosperm surrounding the embryo, and also in several cell layers of the embryo at the torpedo stage (Ibeas et al., 2017). This discovery opens new questions on the mechanisms of iron distribution and accumulation in these seeds, and will be used as the basis for the development of biotechnological strategies to increase total iron content in seeds for human consumption.

A side finding of this work is the broad distribution of Mn in the embryo of C. quinoa hypocotyl (Figure 3B), compared to the restricted location of Mn in the hypocotyl subepidermal tissue of A. thaliana embryos (Kim et al., 2006; Punshon et al., 2012; Schnell Ramos et al., 2013; Eroglu et al., 2017). It has recently been shown that mutations in the genes cax1cax3 (Punshon et al., 2012), mtp8 and vit1 (Eroglu et al., 2017) cause a broad distribution of Mn indicating that they are responsible for this localization. As proposed for iron, this finding will open new strategies for the development of Mn fortified seeds.

In addition to Brassicales, our analyses also showed that at least some Zygophyllales, such as P. chilensis, also have an unusual iron distribution. In this case, iron is distributed inside and outside the cells (apoplast). This iron pool would necessarily be accumulated in a different manner from the vacuolar-located. For instance, there would be no need for particular metal transporters as is the case for Arabidopsis (Lanquar et al., 2005; Kim et al., 2006). It would also be interesting to determine the iron ligands, and how assimilable it is. Further analyses of this and other Zygophyllales species will be carried out to ascertain the extension of this phenotype and to determine its molecular bases.

Regarding the widespread use of Arabidopsis as a Eudicotyledoneae model plant for iron nutrition, our results show that iron distribution in Arabidopsis embryos is not a widely conserved character in Eudicotyledoneae seed embryos and it corresponds to an apomorphic character. Ancestral reconstruction of the phylogeny of angiosperms with Maximum Parsimony or Maximum Likelihood indicated that the embryo of the ancestor of Brassicales, and probably of all Eudicotyledoneae, accumulates iron in several layers of the cortex (Figure 7). The loss of iron in the outer layers of the cortex evolved in the core of Brassicales, whereas the ability to accumulate iron only in endodermis is a derived trait of Arabidopsis. According to Beilstein et al. (2010), the apomorphy described in our study emerged between 120 and 70 Mya. In our opinion, Brassicales seeds could be used to evaluate molecular mechanisms involved in iron accumulation in the cortex cells, using information obtained from other species described in this article.

These results also open new questions on the mechanisms involved in iron accumulation and remobilization in cortex and protodermis cells, during embryogenesis and seed germination, respectively. From an evolutionary point of view, it will be interesting to study which competitive advantage determined the selection of this iron distribution in Brassicales species.

MI, SG-G, JV-P, NN, and HR performed the embryo dissections and Perls/DAB staining. IA, HC-M, and MG-G performed the SμXRF analysis. FP performed the phylogenetic analysis. All authors participated in the writing of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.