Plants evolved complex regulatory mechanisms to cope with changes in the environment, including nutrient availability (Secco et al., 2017). At the molecular level, plants respond to nutritional status by modulating gene expression at multiple levels through a network of transcription factors and via post-translational regulation. Multiple studies have also revealed that changes in chromatin state by histone modification or DNA methylation play important roles in nutrient homeostasis in plants (Secco et al., 2017; Séré and Martin, 2020).

Post-translational modification of histone and DNA methylation lead to transcriptional regulation by altering chromatin packaging and chemical properties of the nucleosome surface, both of which influence association of DNA-binding transcriptional regulators (Berger, 2007). Emerging evidence reveals the importance of chromatin regulation in nutritional homeostasis in plants. For example, phosphate starvation-induced genes are regulated by the histone acetyltransferase GCN5 (Wang T. et al., 2019) and histone deacetylases HDA19 and HDC1 (Chen et al., 2015; Xu et al., 2020). Histone 3 lysine 4 trimethylation (H3K4me3) was also shown to regulate gene expression under phosphate deficiency (Chandrika et al., 2013a,b). In high nitrogen, increased H3K27me3 deposition, and decreased H3K4me3 and H3K36me3 contribute to the repression of the high affinity nitrate transporter gene, AtNRT2.1 (Widiez et al., 2011). H3K27me3 also modulates AtNRT2.1 by limiting its induction under low nitrogen (Bellegarde et al., 2018). Multiple genes involved in sulfate uptake and assimilation are direct targets of histone methylation and acetylation (Huang et al., 2019). In addition, global changes in DNA methylation were observed under phosphate starvation (Yong-Villalobos et al., 2015; Secco et al., 2017), sulfur deficiency (Huang et al., 2016), and zinc deficiency (Chen et al., 2018). Chromatin remodeling genes were differentially expressed upon zinc or iron treatment, implying chromatin-level responses to maintain mineral homeostasis (Darbani et al., 2015). Although chromatin remodeling has not been extensively studied in the context of metal homeostasis, reports increasingly suggest the involvement of histone modification and DNA methylation in regulating iron. This minireview will focus on the current knowledge of chromatin-based regulation of iron homeostasis in plants.

Iron is an essential micronutrient for plant growth and development. Iron is an indispensable cofactor in vital metabolic processes, but improperly regulated iron causes cytotoxicity by facilitating the generation of reactive oxygen species (ROS) (Halliwell and Gutteridge, 1992). Despite being abundant in the soil, iron is not readily accessible for plants, as it is highly insoluble in aerobic conditions at neutral or alkaline pH (Colombo et al., 2014). Iron’s importance as an essential micronutrient with low bioavailability and its potential for toxicity necessitates a tightly regulated system of iron acquisition and regulation in plants. Understanding iron homeostasis is important to answer fundamental biological questions, but also to improve agriculture and human health.

In response to iron deficiency, plants induce iron uptake mechanisms that involve reducing or chelating iron (Connorton et al., 2017; Riaz and Guerinot, 2021). Dicots acquire iron via a reduction-based process known as Strategy I, which involves proton efflux to the rhizosphere by proton ATPases such as AHA2 to solubilize ferric chelates (Santi et al., 2005), coumarin secretion to facilitate iron mobilization (Clemens and Weber, 2016), reduction of ferric chelates to ferrous iron by FERRIC REDUCTASE OXIDASE 2 (FRO2) (Robinson et al., 1999), and ferrous iron import into root epidermal cells by IRON-REGULATED TRANSPORTER 1 (IRT1) (Eide et al., 1996). IRT1, FRO2, and AHA2 co-localize in interactomes, which likely optimize iron uptake (Martín-Barranco et al., 2020). Grasses use a chelation-based process or Strategy II for iron uptake. When iron is limited, phytosiderophores, mugineic acid (MA) and its derivatives, are synthesized (Mori and Nishizawa, 1987; Shojima et al., 1990) and secreted into the rhizosphere by Transporter of Mugineic acid family phytosiderophores 1 (TOM1) to chelate iron (Nozoye et al., 2011). Fe³⁺-phytosiderophore complexes are then transported into the root epidermal cells by the Yellow Stripe (YS) family transporters (Curie et al., 2001). Even though grasses are considered as Strategy II plants, Strategy I is used or its components exist in graminaceous plants (Bughio et al., 2002; Ishimaru et al., 2006; Cheng et al., 2007; Bashir et al., 2011; Li et al., 2016; Kaur et al., 2019; Wairich et al., 2019; Wang M. et al., 2019).

Responses to iron availability are controlled from transcriptional to post-translational levels (Vélez-Bermúdez and Schmidt, 2022). In particular, the complex network of basic helix-loop-helix (bHLH) family transcription factors involved in iron deficiency response has been extensively studied (Gao et al., 2020; Schwarz and Bauer, 2020). In Arabidopsis, FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT)/bHLH29 directly regulates IRT1, FRO2, FIT, and other genes involved in iron uptake under iron deficiency (Colangelo and Guerinot, 2004; Jakoby et al., 2004; Yuan et al., 2005). FIT forms heterodimers with subgroup Ib bHLH transcription factors, bHLH038/39/100/101, to activate FIT-dependent gene expression (Yuan et al., 2008; Wang et al., 2013). FIT also interacts with subgroup IVa bHLHs, triggering the degradation of FIT via the 26S proteasome pathway (Cui et al., 2018). Alongside FIT, POPEYE (PYE)/bHLH47 is another major transcriptional regulator of iron deficiency response in Arabidopsis (Long et al., 2010). PYE is expressed under iron deficiency and negatively regulates its target genes, which include those involved in iron translocation, storage, and assimilation. ILR3/bHLH105 plays a dual role in iron homeostasis; depending on the heterodimer it forms, ILR3 activates PYE expression (Zhang J. et al., 2015) or represses PYE-target genes (Tissot et al., 2019). UPSTREAM REGULATOR OF IRT1 (URI)/bHLH121 directly or indirectly positively regulates multiple iron homeostasis genes of the bHLH network (Kim et al., 2019; Gao et al., 2020; Lei et al., 2020). URI controls nearly half of iron-regulated genes, including both FIT-dependent and independent genes (Kim et al., 2019; Gao et al., 2020). Although URI expression is not iron-regulated, phosphorylation of its protein stabilizes it to form heterodimers with subgroup IVc bHLH transcription factors and activate subgroup Ib bHLH genes under iron deficiency (Kim et al., 2019). Upon iron re-supply, phosphorylated URI is targeted by the E3 ligase BRUTUS (BTS) and subjected to proteasome-mediated degradation. The IRONMAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) peptides also positively regulate iron deficiency response in Arabidopsis (Grillet et al., 2018; Hirayama et al., 2018) by sequestering BTS to prevent degradation of bHLH105/bHLH115 and activate iron uptake (Li et al., 2021).

Responses to iron deficiency in grasses also utilize several bHLH transcription factors (Gao and Dubos, 2021). OsFIT/OsbHLH156 positively regulates Strategy II-related genes such as those involved in MA biosynthesis and also regulates OsIRT1, a Strategy I-related gene (Liang et al., 2020; Wang et al., 2020). OsIRO2 interacts with OsFIT to promote its nuclear localization and positively regulate iron uptake by OsIRT1 (Ogo et al., 2006, 2007; Liang et al., 2020; Wang et al., 2020). OsIRO3/OsbHLH63 represses iron deficiency response possibly via antagonizing OsIRO2 to avoid iron overload by limiting iron uptake (Zheng et al., 2010; Gao and Dubos, 2021).

Each nucleosome consists of an octameric complex of histones subjected to a wide range of post-translational modifications. These modifications are reversible but are controlled by many histone modifying enzymes and play key roles in regulating chromatin structure and transcription (Bannister and Kouzarides, 2011; Zhang T. et al., 2015). Multiple iron homeostasis genes in Arabidopsis have been found to be controlled by histone modifications as discussed in this section.

DNA methylation controls gene expression and contributes to silencing of transposons to maintain genome stability (Law and Jacobsen, 2010; Zhang et al., 2018). In plants, methylation of cytosine occurs in symmetric methylation at CG and CHG, where H represents A, T, or C, and asymmetric methylation at CHH (Matzke and Mosher, 2014; Matzke et al., 2015). While CG and CHG methylations are maintained during DNA replication, CHH methylations are established de novo after DNA replication via RNA-dependent mechanisms and are frequently found between condensed and relaxed chromatin near highly expressed genes (Gent et al., 2013; Martin et al., 2021).

A recent report suggested that CHH DNA methylation modulates iron deficiency response in rice via changing methylation status of genes encoding two major positive regulators of iron deficiency response, OsIRO2 and OsbHLH156 (Sun et al., 2021). In this study, widespread hypermethylation, mainly CHH methylation, was detected in rice roots and shoots grown in iron deficient conditions by mapping the DNA methylome at a single-base resolution. Although little correlation was found between CHH hypermethylation and expression of iron deficiency response genes, OsIRO2 and OsbHLH156 exhibited CHH hypermethylation and their expression increased under iron deficiency. Furthermore, treatment of 5-aza-2-deoxycytidine (Aza), a DNA methylation inhibitor, and the loss of OsDRM2, a key methyltransferase responsible for CHH methylation, resulted in lower expression of OsIRO2 and OsbHLH156, accumulation of less iron, and growth retardment under iron deficiency (Sun et al., 2021). It was speculated that small RNAs might play a critical role as rice acclimates to iron deficiency, as the levels of 24-nt siRNAs increased, whereas transcript levels of canonical RNA-dependent DNA methyltransferases involved in CHH methylation did not change under iron deficient conditions (Sun et al., 2021).

In barley, iron deficiency led to a general reduction of CG methylation, but the overall methylation and demethylation status was not recovered after iron resupply (Bocchini et al., 2015). Further studies are necessary to understand the extent to which DNA methylation or demethylation is maintained upon changes in iron conditions and mechanisms therein.

DNA methylation status was also proposed to be involved in feedback mechanisms between iron status and tolerance to cadmium stress (Fan et al., 2020). Arabidopsis plants exposed to cadmium stress expressed lower levels of the three DNA demethylase genes AtROS1/DML2/DML3 (RDD) and exhibited increased global DNA methylation that resembled the methylation profile of rdd triple mutants (Fan et al., 2020). The rdd mutants were more tolerant against cadmium stress and accumulated more iron in the shoots by expressing higher levels of iron deficiency response genes than wild type. However, inadequate iron supply abolished cadmium tolerance in rdd mutants (Fan et al., 2020).

Increasing evidence has shown that iron homeostasis gene expression is affected by histone modification (Figure 1) and DNA methylation (Figure 2). Such chromatin-based regulation is critical during iron deficiency and allows to fine-tune iron homeostasis in plants. Given that chromatin-based regulation is a dynamic process, it will be important to understand the mechanistic details regarding changes in histone modification or DNA methylation in response to changes in iron status. Research to date has mainly focused on iron deficiency and little is known about the effect of iron overload on chromatin remodeling via histone modification or DNA methylation. Global changes in H3K9me2 and H3K4me3 levels under high iron stress conditions were detected in the proximal root meristem in rice (Polosoro et al., 2019), but further studies are needed to understand the underlying mechanisms and the biological implications. Furthermore, it will be necessary to integrate large scale datasets of various histone modifications, DNA methylation, and the combinatorial effect of different modifications, as well as comparative analyses of transcriptomics and epigenetics of specific cell-types or at a single cell level. Although chromatin-based regulation is an integral part of epigenetics, some chromatin modifications are not heritable or considered epigenetic (Eichten et al., 2014). Thus, transgenerational studies to determine the heritability of chromatin modifications in response to iron will lead to insightful information. Considering the growing evidence that reveal the significance of dynamic adjustment in chromatin structure and subsequent transcriptional changes in response to nutritional status, a clear understanding of chromatin-based iron homeostasis is necessary for a comprehensive understanding of iron homeostasis. Such efforts will contribute insights toward developing crops with improved nutritional profiles and enhanced tolerance to undesirable conditions in the long run.

JS primarily wrote the initial draft of the manuscript. ZY and YW contributed to the manuscript writing and generated the figures. JL and JJ conceived the idea and made final edits. All authors contributed to the article and approved the submitted version.