Fe homeostasis is controlled by a transcriptional cascade involving an intricate network of transcription factors (TFs) regulating the expression of genes encoding proteins involved in Fe uptake, distribution and storage ( Figure 1 ). Fe depletion at the vicinity of plant roots induces the phosphorylation of the basic helix-loop-helix (bHLH) transcription factor bHLH121/UPSTREAM REGULATOR OF IRT1 (URI) and its root cellular relocalization from the central cylinder and the endodermis to the cortex and the epidermis (Gao et al., 2020a). Phosphorylated URI then forms heterodimers with members of the bHLH subgroup IVc (bHLH34, bHLH104, bHLH105/IAA-LEUCINE RESISTANT 3 (ILR3) and bHLH115) (Zhang et al., 2015; Li et al., 2016b; Liang et al., 2017; Kim et al., 2019; Tissot et al., 2019; Gao et al., 2020a; Lei et al., 2020). Cooperatively, these TFs acts as direct transcriptional activators of key genes involved in the Fe regulatory network, including four bHLH Ib genes (bHLH38, bHLH39, bHLH100, bHLH101), POPEYE (PYE/bHLH47), as well as BRUTUS (BTS) and BRUTUS LIKE 1 (BTSL1), which both encode E3 ubiquitin ligases (Kim et al., 2019; Gao et al., 2020a; Lei et al., 2020). In turn, members of the bHLH Ib subgroup associate with bHLH29/FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT) to form heterodimers that promote the expression of the Fe uptake machinery (Colangelo and Guerinot, 2004; Jakoby et al., 2004; Yuan et al., 2008). PYE, a bHLH of the subgroup IVb, interacts with bHLH105/ILR3, to act as a transcriptional repressor of its own expression and of the expression of FERRITIN (FER) genes involved in Fe storage (Long et al., 2010; Samira et al., 2018; Tissot et al., 2019; Akmakjian et al., 2021), which are otherwise induced by bHLH121/URI (Gao et al., 2020b). PYE, likely in interaction with ILR3, also controls Fe redistribution, especially regarding its mobility, through the negative control imposed on the expression of NAS4 (NICOTIANAMINE SYNTHASE 4) involved in the synthesis of nicotianamine (NA), a Fe chelator important for intercellular Fe transport, and on the expression of ZINC-INDUCED FACILITATOR 1 (ZIF1) involved in the vacuolar sequestration of NA in roots (Long et al., 2010; Haydon et al., 2012; Schuler et al., 2012; Tissot et al., 2019). PYE was also found to repress the transcription of bHLH Ib genes (bHLH38, bHLH39, bHLH100, and bHLH101) by directly binding to their promoters, hence triggering the subsequent downregulation of IRT1 and FRO2 (Pu and Liang, 2023). Moreover, two transcription factors of the myeloblastosis (MYB) family, MYB10 and MYB72, cooperate to positively regulate NAS2 and NAS4 expression (Palmer et al., 2013), and the expression of both MYB is induced upon Fe deficiency under the control of heterodimeric complexes FIT/bHLH Ib and might also be dependent on bHLH121/URI (Palmer et al., 2013; Gao et al., 2020a; Lei et al., 2020).

Fine tuning of this complex transcriptional regulatory cascade may be achieved by the Fe-deficiency-dependent phosphorylation of bHLH121/URI and its subsequent phospho-dependent degradation by BTS (Kim et al., 2019). It is noteworthy, however, that BTS expression pattern is restrained to the stele (Selote et al., 2015; Rodríguez-Celma et al., 2019), where BTS would coexist with an unphosphorylated, hence undegradable bHLH121/URI protein (Kim et al., 2019). Furthermore, no interaction was uncovered between bHLH121/URI and BTS or BTSL proteins in yeast two-hybrid assays (Gao et al., 2020a). BTS and its homologs BTSL1 and BTSL2 are all E3 ubiquitin ligases that are able to bind Fe and to adjust their biological function accordingly to negatively regulate Fe homeostasis (Selote et al., 2015; Hindt et al., 2017; Kim et al., 2019). The negative control imposed by BTS and BTSLs results from the proteasomal degradation of their targets, among which are found several bHLHs especially important for Fe homeostasis (e.g. bHLH29/FIT, bHLH104, bHLH105/ILR3 and bHLH115) (Selote et al., 2015; Rodríguez-Celma et al., 2019). BTS and BTSL proteins all possess hemerythrin/HHE Fe-binding domains at their N-terminus (Hindt et al., 2017), and the BTS protein is stabilized in the absence of Fe bound to glutamic acid residues located within HHE domains (Selote et al., 2015). At the transcriptional level, BTS and BTSL genes are upregulated by Fe deficiency at least through the control of three bHLH transcription factors (bHLH105/ILR3, bHLH115 and bHLH121/URI) (Long et al., 2010; Liang et al., 2017; Rodríguez-Celma et al., 2019; Gao et al., 2020a). In contrast, ELONGATED HYPOCOTYL 5 (HY5), a light signaling basic leucine zipper (bZIP) transcription factor, has been identified as a negative regulator of BTS and PYE expression to modulate Fe-deficiency responses (Mankotia et al., 2023). It was recently shown that IRON MAN (IMA) peptides interact with and are ubiquitinated by BTS, which inhibits the degradation of bHLH105 and bHLH115 through binding competition (Li et al., 2021). In the same line of evidence, the interaction of BTSL1 with PYE and bHLHs of the subgroup IVc is attenuated by the peptide IMA1 (Lichtblau et al., 2022). Interestingly, mutation applied to the bHLH121/URI gene leads to an up-regulation of IMA1/2/3 and BTS genes under Fe deficiency (Lei et al., 2020).

Interestingly, crosstalks exist between Fe homeostasis and phytohormone signaling pathways. Indeed, FIT interacts with ETHYLENE INSENSITIVE 3 (EIN3) and ETHYLENE INSENSITIVE 3-LIKE 1 (EIL1) (Lingam et al., 2011). Two components of the jasmonic acid (JA) pathway, MYC2 and JAR1, negatively regulate the expression of FIT and bHLH Ib genes in response to JA (Cui et al., 2018). Moreover, FIT interactions with four IVa bHLHs (bHLH18, bHLH19, bHLH20 and bHLH25) trigger JA-mediated FIT degradation by the 26S proteasome (Cui et al., 2018). FIT, bHLH38 and bHLH39, as well as FRO2, IRT1, expression is also positively regulated by two key enzymes of the gibberellin (GA) biosynthetic pathway (gibberellin 3-oxidase 1 and 2, GA3ox1 and GA3ox2) (Matsuoka et al., 2014). DELLA proteins, which are negatively regulated by bioactive GA, associate with FIT, bHLH38 and bHLH39 to inhibit their activity (Wild et al., 2016).

FIT and Ib bHLH genes, hence their direct targets (at least IRT1 (Schwarz and Bauer, 2020) and putatively NRAMP1, as they are known to be co-regulated (Colangelo and Guerinot, 2004)) are positively regulated by a wide range of metal excess (zinc (Zn²⁺), cobalt (Co²⁺), nickel (Ni²⁺), cadmium (Cd²⁺) and manganese (Mn²⁺)) (Wu et al., 2012; Lešková et al., 2017). The effect of metal excess on the expression of aforementioned genes likely results from competition with Fe uptake as it was demonstrated for cadmium (Wu et al., 2012).

Upon Fe deficiency, many genes mentioned above undergo changes not only at the transcriptional level but also at the post-transcriptional level through modification of their splicing patterns (Lan et al., 2013; Li et al., 2013; Li et al., 2016a; Hsieh et al., 2022). Alternative splicing events occurring upon Fe deficiency might be regulated by many splicing factors, including members of the serine/arginine-rich (SR) family (Zhang et al., 2014; Dong et al., 2018; Fanara et al., 2022). Although an alternatively spliced isoform of IRT1 mRNA retaining an intron has been previously described (Li et al., 2013), no significant changes appear in the IRT1 splicing profile in a wild-type plant upon long- or short-term Fe deficiency (Fanara et al., 2022; Hsieh et al., 2022). Among the genes of the bHLH Ib subgroup, only bHLH100 and bHLH101 undergo differential intron retention (DIR) upon short-term Fe deficiency (Hsieh et al., 2022). While bHLH29/FIT intensively undergoes differential donor or acceptor (DDA) splice sites events (Hsieh et al., 2022), changes in the NRAMP1 splicing profile occur through both DIR (Lan et al., 2013; Li et al., 2013) and DDA (Lan et al., 2013; Li et al., 2016a). DDA events define new borders between exon and intron at the 5’ and 3’ ends of the intron, respectively, and might result in frameshifts and subsequent premature termination codons. Alternatively, if in-frame, DDA could modify the length of exonic regions (English et al., 2010). Alternative splicing therefore has the potential to partially modify important domain of a protein, hence modifying its interaction network, function, stability, or even its intracellular localization (English et al., 2010; Kalyna et al., 2012; Drechsel et al., 2013; Remy et al., 2013).

Membrane proteins are synthesized in the endoplasmic reticulum (ER) (High and Laird, 1997), and use well-characterized secretion signals and pathways to traffic in the cell to their destination (Peer, 2011; Chung et al., 2016). Exit from the ER requires the direct interaction with the coat protein complex II (COPII) using specific motifs, which allows their transport to the Golgi apparatus (Barlowe et al., 1994; Matheson et al., 2006; Lord et al., 2013). Then, membrane proteins follow the secretory pathway through the trans-Golgi network (TGN) and reach their final destination using various vesicles such as clathrin-coated vesicles or exocysts (Matheson et al., 2006; Peer, 2011; Chung et al., 2016). The mechanisms by which IRT1 and NRAMP1 reach the plasma membrane is still elusive and lacks experimental evidence. However, one of the first characterized ER export motifs, the diacidic motif (D/E)X(D/E), is present in both transporters (Hanton et al., 2005). NRAMP1 harbors a diacidic motif (DVD) in its cytoplasmic C-terminal part, as many others membrane proteins, and IRT1 possesses the diacidic motif (EDD) in its largest cytoplasmic loop. Besides, mutation of this motif in IRT1 from Malus xiaojinesis leads to its retention into the ER (Tan et al., 2018). In addition, two of the five COPII core components, SEC13a and SEC31b, were recently identified in the Arabidopsis IRT1 interactome (Matheson et al., 2006; Lord et al., 2013; Martìn-Barranco et al., 2020). Altogether, these findings suggest that IRT1 is exported from the ER to the Golgi apparatus using the COPII machinery. Then, during the post-Golgi trafficking, the CHOLINE TRANSPORTER-LIKE 1 (CTL1) plays an essential role to allow the delivery of NRAMP1 and probably IRT1 at the plasma membrane (Gao et al., 2017).

Interestingly, both transporters have low Fe specificity allowing the entry, in plant cells, of others divalent metals (hereafter named non-Fe metals), such as Zn²⁺, Mn²⁺, Co²⁺, and Cd²⁺ (Vert et al., 2002; Cailliatte et al., 2010; Barberon et al., 2011). In order to fine tune Fe acquisition and hence limit toxicity of their non-Fe metal substrates, both transporters are regulated at the post-translational level leading to a complex intracellular trafficking ( Figure 2 ) (Barberon et al., 2011; Barberon et al., 2014; Agorio et al., 2017; Dubeaux et al., 2018; Castaings et al., 2021; Spielmann et al., 2022). Besides Fe transport, NRAMP1 is also essential for manganese uptake (Curie et al., 2000; Cailliatte et al., 2010; Castaings et al., 2016). Indeed, manganese availability impacts NRAMP1 subcellular localization. In control condition, NRAMP1 is shared between plasma membrane and endosomal vesicles of the trans-Golgi network/early endosome (TGN/EE) and the late endosome/multivesicular body (LE/MVB) (Agorio et al., 2017; Castaings et al., 2021). Increasing manganese availability gradually reduces the amount of NRAMP1 at the plasma membrane while enhancing its presence in the endosomal fraction (Castaings et al., 2021). This manganese-induced NRAMP1 endocytosis involves the clathrin-mediated endocytic pathway and occurs after phosphorylation of NRAMP1 (Castaings et al., 2021). The increase of intracellular manganese concentration seems to induce an intracellular transient calcium signal which is perceived by two calcium sensors, the CALCINEURIN-B-LIKE 1 (CBL1) and CALCINEURIN-B-LIKE 9 (CBL9). CBL1/9 interact with and activate the CBL-INTERACTING PROTEIN KINASE 23 (CIPK23) which phosphorylates NRAMP1 at serine 20 (S20), thereby inducing endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to manganese toxicity. Indeed, in cipk23 and cbl1/cbl9 mutant backgrounds, NRAMP1 manganese-mediated endocytosis is abolished (Zhang et al., 2023b). Additionally, a nonphosphorylatable NRAMP1 mutant on S20 is blocked at the plasma membrane unlike the corresponding phosphomimic variant which is constitutively endocytosed, thus suggesting that phosphorylation is a pivotal step for NRAMP1 trafficking (Castaings et al., 2021). Finally, NRAMP1 undergoes vacuolar degradation through an unclear process depending on the PLECKSTRIN HOMOLOGY (PH) DOMAIN-CONTAINING PROTEIN 1 (PH1), but independent of manganese availability (Agorio et al., 2017; Castaings et al., 2021). Even if perturbation of NRAMP1 endocytosis was found to cause manganese toxicity (Castaings et al., 2021), most factors and mechanisms driving NRAMP1 trafficking and degradation remain to be identified and characterized.

In the absence of Fe and non-Fe metals, IRT1 is mainly localized at the plasma membrane of root epidermal cells in a soil-facing polar fashion to optimize Fe uptake (Barberon et al., 2014; Dubeaux et al., 2018). When plants are exposed to non-Fe metals, IRT1 is subjected to a rapid and complex post-translational regulation to limit accumulation and toxicity of non-Fe metals. IRT1 acts as a transceptor, combining transporter and receptor activities, enabling sensing of intracellular metal status and regulation of its cell surface levels (Cointry and Vert, 2019). Increasing the concentration of non-Fe metals gradually removes IRT1 from the cell surface, allowing IRT1 to traffic through endosomal compartments on its way to vacuolar degradation (Dubeaux et al., 2018). Thanks to a histidine-rich motif located in the largest cytosolic loop (thereafter called IRT1c), IRT1 directly binds non-Fe metals (Dubeaux et al., 2018; Spielmann et al., 2022). This interaction triggers the recruitment of the CIPK23 kinase that phosphorylates IRT1c and thus creates a docking site for the E3 ubiquitin ligase IRON DEGRADATION FACTOR 1 (IDF1) (Shin et al., 2013; Dubeaux et al., 2018). Through the cooperation with two E2 UBIQUITIN CONJUGATING ENZYMES 35 and 36 (UBC35 and UBC36), IDF1 decorates two IRT1c lysine residues (K159 and K174) with K63-linked polyubiquitin chains (Shin et al., 2013; Dubeaux et al., 2018; Spielmann et al., 2022). IRT1 is then sorted toward late endosomes (LEs) and the vacuole for degradation, but the exact underlying mechanisms remain unclear (Barberon et al., 2011; Dubeaux et al., 2018; Spielmann et al., 2022). Polyubiquitinated IRT1 is probably recognized by the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT (ESCRT) complex to be sent to the vacuole for degradation unless recycled (Dubeaux and Vert, 2017). In fact, two recycling mechanisms have been identified to exit the route to the vacuole (Barberon et al., 2014; Ivanov et al., 2014). One of these pathway preventing IRT1 degradation involves the SORTING NEXIN 1 (SNX1) protein which partly co-localise with IRT1 in TGN/EE and to a lesser extent in LE. A snx1 loss-of-function plant displays lower IRT1 levels than wild-type plants due to enhanced degradation rate. This suggests a role for SNX1 in the recycling of IRT1 (Ivanov et al., 2014). Secondly, IRT1 interacts with the phosphatidylinositol-3-phosphate-binding protein FYVE1 in LE, which alters its fate. Indeed, plants with enhanced expression of FYVE1 not only show an accumulation of IRT1 at the plasma membrane, but also display a loss of IRT1 lateral polarity (Barberon et al., 2014). Considering that FYVE1 is part of the ESCRT complex and mediates MVB sorting (Gao et al., 2014), the mechanisms by which FYVE1 overexpression reroutes IRT1 to the plasma membrane are still unclear.

IRT1 was also recently demonstrated to be degraded via the 26S proteasome and autophagy pathways. Indeed, IRT1 directly interacts with the plasma membrane-localized RING-type E3 ubiquitin ligase ARABIDOPSIS TOXICOS EN LEVADURA 31 (ATL31), which enhances IRT1 ubiquitination and leads to both proteasome- and autophagy-dependent degradation in response to cadmium stress. In addition, the WRKY33 transcription factor directly activates ATL31 expression in response to cadmium stress, suggesting a WRKY33-ATL31-IRT1 regulatory module involved in cadmium-specific plant tolerance (Zhang et al., 2023a). It remains to be determined how the proteasome may convey the degradation of a highly hydrophobic membrane protein like IRT1.

Finally, IRT1 post-translational regulatory mechanisms have been proposed to modulate Fe uptake. The peripheral membrane protein ENHANCED BENDING 1 (EHB1), a calcium-binding protein, acts as an Fe uptake inhibitor even during Fe starvation. EHB1 binds to IRT1c and thus interacts with IRT1 in a calcium-dependent manner, reducing Fe acquisition by a still unknown process. The IRT1-EHB1 interaction might represent a rapid mechanism to switch off Fe import once its optimal intracellular concentration has been reached, avoiding Fe overaccumulation and toxicity (Khan et al., 2019). The peripheral plasma membrane SEC14-Golgi dynamics (SEC14-GOLD) protein PATELLIN 2 (PATL2) also binds IRT1c to reduce membrane oxidative damage during Fe import via IRT1 (Hornbergs et al., 2023). In contrast, Fe deficiency leads to phosphorylation of serine 149 (S149) in IRT1c by the CALCIUM-DEPENDENT PROTEIN KINASE 21 and 23 (CPK21 and CPK23) calcium-regulated kinases to increase IRT1 transport activity to optimize Fe uptake (Wang et al., 2023). In response to manganese depletion, these two kinases also catalyze the phosphorylation of NRAMP1 at threonine 498 (T498), leading to NRAMP1 activation and optimal manganese uptake (Fu et al., 2022).