VIT1-dependent Fe distribution in seeds is conserved in dicots

One third of the people suffer from iron (Fe) Fe deficiency. An underlying factor for this malnutrition is insufficient Fe intake from the diet. A major part of the human diet includes seeds of staple crops, which contain Fe that is poorly bioavailable. One reason for the low bioavailability is these seeds store Fe in cellular compartments that also contain antinutrients, such as phytate. Thus, several studies focused on decreasing phytate concentrations. As an alternative approach to increase bioavailable Fe, Fe reserves might be directed to cellular compartments such as plastids that are free of phytate. Previous studies indicated that Fe reserves can be relocalized inside the seed to the desired compartment by genetic modification, provided that a suitable iron transporter protein is used. However, to the best of our knowledge, a Fe transporter localizing to plastids have not been identified in seeds to date. To discover novel Fe transporters, we screened Fe patterns in seeds of distinct plant lineages, hypothesizing Fe hyperaccumulating sites would indicate Fe transporter presence. To this end, metal localizations in seeds of more than twenty species were investigated using histochemical or X-ray based techniques. Results showed that in Rosids, the largest clade of eudicots, Fe reserves were primarily confined in the embryo part of the seeds. Furthermore, inside the embryos, Fe was enriched in the endodermal cell layer, a well-known feature that is mediated by vacuolar Fe transporter, VIT1 in model plant Arabidopsis thaliana. This enrichment was well conserved in and beyond Rosid species. Finally, a few seeds showed novel Fe patterns, including Carica papaya which concentrated large Fe reserves exclusively in plastids called amyloplasts. Generally, Fe stored in amyloplast is considered bioavailable. Taken together, this study suggests dicot seeds store Fe mainly in the embryo, with a VIT1-dependent enrichment in its endodermal cell layer and indicate Carica papaya possess a strong Fe transporter at the plastid membrane. Once it is identified that might be useful in biofortification, as a novel tool to shift Fe to compartments where it is more bioavailable.

H 2 O 2 , and 0.005% (w/v) CoCl 2 (intensification solution). The reaction was stopped by rinsing   and outer), both of which exhibited the same Fe distribution pattern (Fig.1B, D). Despite the 1 9 5 similarities in these patterns, variations between species were also observed. In contrast to 1 9 6 Arabidopsis thaliana (Fig.1A), Fe enriched region of Brassica napus was not confined to a 1 9 7 single cell layer in the hypocotyl (Fig.1C). Furthermore, Alyssum sibiricum showed two 1 9 8 adjacent Fe enriched circles instead of one in its hypocotyl (Fig.1D). Taken together, results showed that not only A. thaliana, but also other Brassicaceae species store main Fe reserves in of either cotyledon or hypocotyl, which is the typical pattern that was seen in Brassicaceae either Perls alone (for Fe rich samples) or with DAB intensification (for those that produce 2 0 7 low signal, i.e., low Fe concentration) to reach a balance in staining intensity. In Limnanthes 2 0 8 douglasii embryos, at the first glance, not only endodermal cells but most of the others were 2 0 9 stained ( Figure 2A). However, closer examination around provascular bundles of cotyledons 2 1 0 and comparison of these cells with nearby cells revealed that single cell layer around the 2 1 1 strands (i.e., endodermis) were slightly enriched with Fe ( Figure 2B). In Capparis spinosa, 2 1 2 Perls staining without DAB amplification revealed that Fe was accumulated close to the in several cell layers surrounding the provasculature including endodermis (Fig. 2E). Therefore, Fe enriched endodermis that was initially observed in A. thaliana, is conserved in 2 1 7 plants at least in order level. Next step was to further pursue the Fe distribution pattern in orders distinct from Brassicales.

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Brassicales and sixteen other orders together constitute Rosids (Chase et al., 2016). Random 2 2 2 species belonging to diverse orders under Rosids were collected (Table 1). Gossypium seed. Likewise, Eucalyptus elata also showed Fe enrichment in the endodermis. However, this 2 2 6 enrichment was evident in cotyledons but failed to be discerned in the hypocotyl, where 2 2 7 cortical cells were already heavily stained (Fig.3B). Two species that belong to the Fagales  In Arabidopsis thaliana seed, Fe enrichment in endodermis is strictly dependent on a 2 3 6 functional VIT1 protein (Kim et al., 2006). We failed to observe Fe enriched endodermis in 2 3 7 some species, indicating they may not have a functional VIT1. In order to test how well VIT1  Next, we assessed conservation of VIT1 protein sequence in species that were used in Fe staple crops are largely unavailable to human because it is associated with antinutrients such 3 0 0 as phytate. Directing Fe from phytate containing vacuoles to phytate-free compartments, in 3 0 1 particular to plastids; where Fe is rather bound to ferritin, producing a more bioavailable 3 0 2 complex, might be a beneficial strategy. This strategy requires identification of a plastid-3 0 3 localized strong Fe transporter. To find such transporters, using Perls/DAB staining and X-ray 3 0 4 based methods, many seeds that belong to distinct plant lineages were screened for Fe 3 0 5 accumulating plastids. This screening revealed that in Carica papaya seed, large Fe reserves 3 0 6 exclusively localize to amyloplasts.  transporter responsible for this plastid-localized Fe distribution.

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In this study, analysis of Fe distribution in seeds also reveal the general Fe distribution 3 2 3 patterns in species of Rosid seeds. Fe is mostly stored in the embryo part of the seeds (Fig 1-3, embryo occupying more than half of the total seed volume. In contrast, in later appeared 3 3 0 species (i.e., Rosids) endosperm shrinks as the seed develops and embryo occupies most of 3 3 1 the seed. Since endosperm is a nutritive tissue, as it degrades, nutrient storage function must 3 3 2 be taken over by the embryo itself. Therefore, we suggest metal accumulation function of the trend. In this process, metal transporters that would create sinks in the embryo might be Besides the general trends among seeds, exceptional distribution of Fe was also detected. seed, as the mature coat is dead while the endosperm is sterile. Although Fe that is trapped in imaging is not a feasible option. Note that zinc is not trapped in the seed coat, which might be Supp. Fig.4). Interestingly, X-ray fluorescence showed Fe in the endosperm colocalizes with 3 5 7 phosphorus (P) (Fig. 7A). Localization of P generally mirrors phytate distribution in seeds,   We showed that Fe accumulation exhibits hot spots in embryos, which correspond to the 3 6 3 endodermis (Fig. 8). Among other Fe enriched regions, endodermis was detected as the only 3 6 4 conserved one among distinct plant lineages (Fig. 8). In Arabidopsis thaliana, Fe enriched  Therefore, we assumed Fe-enriched endodermis in species other than Arabidopsis thaliana 3 6 7 was also due to the presence of VIT1 homologs in those species. In few species, Fe enriched 2D, also see Ibeas et al. (2017)). Therefore, the question arises whether VIT1 can localize to 3 7 0 cells other than endodermis or is it strictly cell-type specific. As elegantly shown by restricted to a single cell layer or not, the typical ring-shaped Fe localization around 3 7 4 provascular strands is always due to the expression of VIT1.

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Although VIT1-mediated Fe enrichment was conserved even in distinct orders, few species 3 7 6 did not exhibit this phenotype (Fig. 5-8). Among those seeds, endodermis in papaya embryo 3 7 7 was devoid of any staining, while, in all the rest, staining in endodermis were not noticeably higher. This may pose the question whether VIT1 has been lost in these species during the  Likewise, when a more preferential Fe transporter is present, VIT1's impact on Fe distribution or Perls/DAB staining (Fig. 1A) and the signal from the rest of the cells is unnoticeable.

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Nevertheless, the rest of the cells in the embryo account for half of the total Fe in the seed, as its associated phenotype is well conserved despite some seeds show alternative Fe hotspots. Rosids is a huge lineage including 80000 species belonging to 147 families, making more 3 9 7 than a third of all angiosperms (Hedges and Kumar, 2009;Soltis, 2005). The current study 3 9 8 shows seeds store Fe mainly in embryo in Rosid species. This Fe is not equally distributed but   Dr. Emre Cilden (Hacettepe University, Ankara, Turkey) for sharing seed stocks with us. We   Authors declare no conflict of interest.        Evolution 56, 2112-2125.  Online 14, 1311-1327. Rev. Food Sci. Food Saf. 13, 329-346. phytic acid content of maize and soybean seeds. Nat. Biotechnol. 25, 930-937.