Iron plays an essential role in cells, due to its role as a cofactor in many proteins. Although iron is one of the most abundant elements in the earth’s crust, it is the least bioavailable, especially in calcareous or alkaline soils (Marschner, 1995). In plants, iron is mostly required in mitochondria and chloroplasts, where it is mainly used in the electron transport chain of respiration and photosynthesis, respectively (Jeong and Guerinot, 2009). Iron deficiency in plants causes reduction of vegetative growth and yield loss in crops (Abadía et al., 2011). In humans, iron deficiency is one of the leading causes of anemia worldwide, affecting about one billion people, mainly pregnant women and preschool children of underdeveloped countries (World Health Organization).¹ One of the world health organization objectives of global nutrition for 2025 is to reduce anemia in women of childbearing age by 50%. In pursuance of this aim, it is proposed to increase the iron intake through the biofortification of crops and food (Murgia et al., 2012).

Seeds are a significant iron sink in plants and they are one of the primary source of iron for animal nutrition. During plant evolution and also by anthropogenic causes, iron accumulation and iron distribution in seeds has been modified (DeFries et al., 2015; Ibeas et al., 2019; Vidal Elgueta et al., 2021). For humans, grains contribute half of the dietary iron (Guerinot and Yi, 1994; Trumbo et al., 2001). During maturation stages of seed development, embryos are loaded with iron (Ravet et al., 2009; Roschzttardtz et al., 2009). Little information is available about the mechanisms involved in iron acquisition and accumulation by embryonic cells. Grillet et al. (2014) reported that ascorbate efflux from Arabidopsis thaliana embryos could be necessary for the reduction of Fe³⁺ previous to its uptake by the embryo. Citrate, a weak organic acid known to be one of the main iron ligands, has been suggested to play a role in iron uptake by germinating Arabidopsis thaliana embryos (Roschzttardtz et al., 2011).

In seeds, iron is stored in vacuoles, unlike vegetative tissues where the main pool of iron is found in other subcellular compartments including plastids (leaf mesophyll, pollen grains) and parietal space (root vascular cylinder) (Roschzttardtz et al., 2009, 2013). Two methods have been widely used to visualize iron in the seed: synchrotron radiation micro X-ray fluorescence (SμXRF), and a histochemical method based on the Perls/DAB dye (Roschzttardtz et al., 2009). These methods have shown that iron accumulates in the vacuoles of the endodermis cell layer during seed maturation (Kim et al., 2006; Roschzttardtz et al., 2009; Mary et al., 2015).

One of the major transporters allowing the entrance of iron into the endodermal vacuoles is the tonoplast-localized VACUOLAR IRON TRANSPORTER 1 (VIT1) (Kim et al., 2006). So far, VIT1 is the only player described to be involved in iron acquisition and distribution during the maturation of Arabidopsis embryos. VIT1 transcripts are transiently accumulated during embryo maturation and decrease at the mature stage (Kim et al., 2006). In vit1 mutant embryos, iron is mislocalized into the subepidermal cells of the cotyledons (Kim et al., 2006; Roschzttardtz et al., 2009; Chu et al., 2017; Eroglu et al., 2017; Höller et al., 2022). Also, vit1 grows poorly compared to wild type seeds in alkaline soil where iron is scarcely available, indicating that proper iron distribution in the embryo is crucial for iron metabolism during germination (Kim et al., 2006; Roschzttardtz et al., 2009; Mary et al., 2015). During seed germination, transcripts encoding NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN 3 and 4 (NRAMP3 and NRAMP4) transporters accumulate. NRAMP3 and NRAMP4 are responsible for iron remobilization from the vacuoles of endodermis cells during post-germinative growth (Lanquar et al., 2005). Consequently, nramp3 nramp4 double mutant plants cannot remobilize the iron accumulated in the embryo, thereby slowing down post-germinative development (Lanquar et al., 2005).

Ferritins are important players in iron homeostasis. Ferritins form multimeric complexes composed of 24 subunits organized in a sphere shape, and each complex can store up to 4.500 atoms of iron in its central cavity (Guerinot and Yi, 1994; Ravet et al., 2009). By complexing iron, ferritins play an essential role in reducing oxidative damage in the cell. Importantly, iron excess increases ferritin abundance in roots and shoots (Reyt et al., 2015). Therefore, the expression of FERRITIN genes is used as a proxy for iron availability in the cell. No reports have been published about the genetic control of FERRITIN gene expression during seed development. In Arabidopsis, four genes encode FERRITIN proteins, and AtFER2 is the only one expressed in the late maturation stages of seed development, during iron loading. AtFER2 protein has been detected in desiccated seeds, while AtFER1, AtFER3, and AtFER4 proteins are detected before the maturation stage of seed development and after germination. Interestingly, Atfer2 mutant seeds have a low germination rate. Also, Atfer2 seeds are more susceptible to pro-oxidant agents during seed germination and early post-germinative development (Ravet et al., 2009), confirming the role of AtFER2 in cell protection against oxidative stress.

Like proteins, sugars, and lipids, iron is stored in seed for later utilization by the germinating plantlet (Lanquar et al., 2005; Kim et al., 2006; Roschzttardtz et al., 2009). If the overall genetic regulation of iron uptake is quite well described and understood (Gao et al., 2019; Gao and Dubos, 2021), little information for seed iron loading and distribution is available (Roschzttardtz et al., 2020; Sun et al., 2021). Several transcription factors have been involved in the regulation of gene expression during seed development. Among them, B3 transcription factors, like FUSCA3 (FUS3), LEAFY COTYLEDON2 (LEC2), and ABSCISIC ACID INSENSITIVE3 (ABI3) play a pivotal role during embryo maturation (Ooms et al., 1993; Meinke et al., 1994; Kroj et al., 2003; To et al., 2006; Santos-Mendoza et al., 2008; Carbonero et al., 2017). Mutants for these transcription factors accumulate less seed storage compounds (sugars, lipids, and proteins). Mutants of B3 transcription factors show different phenotypes, such as: ectopic trichomes (lec2), accumulation of anthocyanins in cotyledons (fus3 and lec2), the inability of degrading chlorophyll (abi3 and lec2), and intolerance to desiccation (abi3 and fus3) (To et al., 2006).

In this work, we sought to determine whether the FUS3, LEC2, and ABI3 transcription factors control the pattern of iron stores in seed. To that aim, we studied how seed iron homeostasis is affected in B3 transcription factors mutants using histological and molecular approaches. We determined that iron distribution is modified in abi3, lec2, and fus3 embryo mutants. However, total iron content in dry mutant seeds showed no difference compared to wild type. This result suggests, at least in what was evaluated by the effect of the studied mutants, that the embryo does not control how much iron is delivered in seed during seed development. At the molecular level, we showed that genes encoding ferritins are misregulated in mutant seeds. Finally, we propose a genetic model involving B3 transcription factors ABI3, LEC2, and FUS3 in iron homeostasis in Arabidopsis embryos.

Using Arabidopsis and other dicot model plants like Brassica napus and Chenopodium quinoa, it has been shown that iron accumulates in embryo during seed maturation (Roschzttardtz et al., 2009; Ibeas et al., 2017, 2019). However, how this process is regulated genetically remains elusive. As a gene candidate approach and using Arabidopsis thaliana as a plant model, we focused our study on mutants of the B3 transcription factors family like LEC2, FUS3, and ABI3, known as being master regulators of embryo maturation gene expression and also regulators of the setting up of the seed reserves (Santos-Mendoza et al., 2008). Using Perls/DAB staining we were able to determine the pattern of iron accumulation in mutant embryos (Figures 1–4). Iron distribution in the different embryo mutants showed dramatic differences compared with that of WT embryos, indicating that LEC2, FUS3, and ABI3 transcription factors play a crucial role in iron distribution in embryos. Interestingly, total iron content in mutant seeds was not affected (Figures 1H, 2H, 3H). Additional and more quantitative analyses should be carried out in order to determine where iron accumulates in the mutant seeds in non-embryonic tissues. The fact that total iron was not modified in the mutant seeds suggests that iron could be accumulated in others seed structures like integuments. In the case of lec2-1, our experimental approach does not allow determining if the hypocotyl endodermal iron pool is larger in the mutant compared to the WT. Unlike other non-mineral nutrients that are synthesized and accumulated in the seeds, iron is transported into the seed from the maternal tissue, suggesting that the maternal tissue determines the amount of iron delivered to the seed independently of where iron accumulates in the embryo. Using synchrotron X-ray fluorescence microtomography, Kim et al. (2006) showed that in the embryo of the vit1 mutant, iron is mislocalized. Interestingly, total iron content in vit1 seeds is unchanged, as in mutants of the B3 transcription factors. All these results strongly indicate that ABI3, LEC2, and FUS3 transcription factors regulate iron distribution in the embryo whereas they do not control iron loading from maternal tissues. Nevertheless, how B3 transcription factors modulate iron distribution in embryos is still to be determined. Whether this is by directly modulating the expression of iron transporters is an opened question. Conversely, defects in iron uptake, as is the case for the bhlh121 loss-of-function mutants (for the bHLH121 master transcriptional regulator of iron homeostasis and AtFER1, AtFER3, and AtFER4 expression), leads to decrease in seed iron content without affecting iron distribution (Gao et al., 2020a,b). Recently, it has been described in Arabidopsis that INO, a member of the YABBY transcription factors family, regulates negatively seed iron loading. A reduced expression of INO increased iron accumulation in the embryo without changing its distribution pattern (Sun et al., 2021).

Perls/DAB staining allowed us to observe where iron is localized in the mutant embryos at the subcellular level. Iron was detected in dots resembling iron-ferritin complexes (Divol et al., 2013; Roschzttardtz et al., 2013; Figures 3C,D and Supplementary Figure 1). Iron-ferritin complexes have been detected in seed from several plant species, but in Arabidopsis no more than 5% of total iron in seed is found associated to ferritin (Briat et al., 2010; Moore et al., 2018). Ferritin-less mature seeds, obtained by mutating the AtFER2 gene, have identical iron content than WT seeds (Ravet et al., 2009; Briat et al., 2010). We confirmed by different approaches that ferritins are misregulated in lec2-1, fus3-3, and abi3-6 seeds at the transcript and protein levels (Figure 5). The qRT-PCR analysis suggested that AtFER1 could be the major ferritin form accumulated in the lec2-1, fus3-3, and abi3-6 mutants seeds, but we could not confirm this at the protein level because anti-ferritin immunodetection does not allow discriminating between the four types of ferritin polypeptides in Arabidopsis. It will be interesting to determine spatial expression pattern of specific ferritins and other relevant genes involved in iron transport, on the embryo tissues and its correlation with the iron accumulation. Ferritins are deregulated at the transcript and the protein levels in lec2 mutant seeds (Figure 5); however, iron-ferritin complexes were not detected in the lec2 mutant embryo by Perls/DAB staining (Figure 2). We could speculate that: (i) iron is not available to form complexes with ferritins in lec2 embryos, implying that vacuoles of endodermis cells in hypocotyl, expressing VIT1 transporter, are efficiently depleting iron from all cell embryos, hence preventing iron-ferritin complex formation; or (ii) that ferritins are accumulating in other seed structures like integuments. In both cases, it will be interesting to perform a ferritin immunodetection on embryo sections in order to decipher where ferritins are accumulated in lec2 mutant and WT seeds. From a genetic point of view, our results indicate that LEC2, FUS3, and ABI3 are negative regulators of AtFER1, FUS3, and LEC2 are positive regulators of AtFER2, LEC2 is a negative regulator of AtFER3, and lastly, AtFER4 is negatively regulated by LEC2 and positively regulated by FUS3 and ABI3 (Figures 5, 6). Unlike FUS3 and ABI3, that seem to regulate iron accumulation in the whole embryo, LEC2 is controlling iron accumulation in cotyledons (Figures 1–4, 6). In order to determine whether B3 transcription factors directly control the expression of FERRITIN genes, we generated a gene regulatory network (GRN) composed of genes whose expression is differentially expressed in abi3, fus3, or lec2 mutants (Supplementary Table 2). We searched for possible transcription factor-target interaction data between differentially expressed genes in the CisBP database (Weirauch et al., 2014), the Plant Cistrome Database (O’Malley et al., 2016) and in the Arabidopsis Gene Regulatory Interaction Server (Yilmaz et al., 2011). To narrow our GRN to genes that may be interacting in the context of seed development, we calculated coexpression between transcription factor-target pairs based on transcriptomics data from developing seeds obtained from AtGenExpress (Schmid et al., 2005). The resulting GRN contains 267 nodes and 762 edges (Supplementary Table 3). We found evidence for direct interaction of B3 transcription factors for 158 genes, of which 86 have an associated GO term related to iron according to the Thalemine database (Supplementary Table 4). Concerning B3 regulation of FERRITIN genes, we found evidence for direct control of FER2 by ABI3 and LEC2 and of FER4 by ABI3 and FUS3 (Supplementary Figure 3). In accordance with these findings, using the Eukaryotic Promoter Database (Dreos et al., 2017),² we found ABI3 and LEC2 binding motifs in the FER2 promoter (at −1668, −1667, −1549, and −557 for ABI3 and −1822, −1821, −1666, −535 for LEC2, considering −2000 to 100 bp from the FER2 transcriptional start site) and ABI3 and FUS3 binding motifs in the FER4 promoter (at −1138, −1139, −1219, −1220, −1741, −1750 for ABI3 and −1138, −1139, −1219, −1220, −1587, −1588, −1750, −1751 for FUS3, considering −2000 to 100 bp from the FER4 transcriptional start site). In the case of FER1 and FER3, no evidence for potential direct binding of B3 transcription factors was found; however, we were able to find an indirect potential regulation of FER1 and FER3 via the ethylene-related transcription factors EIL1, EIL3, and EIN3 that act downstream LEC2 and FUS3 (Supplementary Figure 3). Besides FER1 and FER3, these ethylene-related transcription factors also target FER2 and FER4 either directly or by indirect control by transcription factors WRKY9 and TCP24 (Supplementary Figure 3). Finally, transcriptomic public data shows that other iron-related genes such as VIT1 and MTP8 iron transporters are misregulated in some of the B3 transcription factor seed mutants analyzed in this article (Supplementary Figure 4). For instance, VIT1 is downregulated in fus3-3 mutant, which could help explain the loss of iron accumulation in the vacuoles of the endodermis in this mutant. Further studies should be performed in order to describe how FERRITIN genes are regulated during seed development, and the mechanism by which LEC2, FUS3, and ABI3 regulate their expression.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

SG-G and HR conceived, designed the experiments, and wrote the manuscript. SG-G, MS, PB-G, FeG, GC, EV, and HR performed the experiments. SG-G, EV, FrG, CD, CC, and HR analyzed the data. CD, CC, and HR contributed to reagents and materials. All authors contributed to the article and approved the submitted version.

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.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.