Epigenetic regulation of iron homeostasis in Arabidopsis

Abstract

Iron (Fe) is one of the most important microelement required for plant growth and development because of its unique property of catalyzing oxidation/reduction reactions. Iron deficiency impairs fundamental processes which could lead to a decrease in chlorophyll production and pollen fertility, thus influencing crop productivity and quality. However, iron in excess is toxic to the cell and is harmful to the plant. To exactly control the iron content in all tissues, plants have evolved many strategies to regulate iron homeostasis, which refers to 2 successive steps: iron uptake at the root surface, and iron distribution in vivo. In the last decades, a number of transporters and regulatory factors involved in this process have been isolated and identified. To cope with the complicated flexible environmental conditions, plants apply diverse mechanisms to regulate the expression and activity of these components. One of the most important mechanisms is epigenetic regulation of iron homeostasis. This review has been presented to provide an update on the information supporting the involvement of histone modifications in iron homeostasis and possible future course of the field.

Iron Uptake in Arabidopsis

Several components involved in iron uptake have been characterized in the model plant Arabidopsis thaliana. Of note, Iron Regulated Transporter1 (IRT1) and Ferric Reductase Oxidase2 (FRO2) are 2 critical genes responsible for iron uptake. IRT1 is the major transporter necessary for high-affinity iron uptake from the soil and is a key player in the regulation of plant iron homeostasis, as attested to by the severe chlorosis and lethality of irt1 mutants.^1-3 Encoding a ferric chelate reductase, FRO2 is iron-deficiency inducible and functions in the reduction of Fe³⁺ at the root surface.^4,5 In addition, a number of transcription factors have been identified to modulate the iron uptake process. FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) was first identified benefiting from the research of its ortholog, the tomato FER.⁶ In the absence of extra iron, the fit-1 null mutation is lethal at the seedling stage, and approximately half of the Fe deficiency-inducible genes are deregulated in fit-1 roots, which is iron deficient.⁷ However, simply constitutive expression of FIT could not promote the expression of its targets, FRO2 and IRT1, since their induction depends upon the dimerization of FIT with other 4 Ib subgroup bHLH proteins, bHLH38, bHLH39, bHLH100, and bHLH101, whose expression is drastically induced by iron deficiency and repressed by iron overload.⁸ These 4 Ib bHLH genes are induced independently from FIT, and the regulatory mechanism how FIT and Ib subgroup bHLH transcription factors coordinate remains elusive. Recently, bHLH104, belonging to the IVc subgroup of bHLH family, was characterized as a key component positively regulating iron deficiency responses. The bHLH104 interacted with IAA-LEUCINE RESISTANT3 (ILR3) and the complex could directly bind to the promoters of Ib subgroup bHLH genes and POPEYE (PYE), thus regulating the iron uptake process.⁹

Iron Distribution in Arabidopsis

Iron distribution to different organs and tissues is conducted by divalent metal chelators, such as nicotianamine (NA) or citrate, which could cross membranes via transporters from the Yellow-stripe Like (YSL) family or the protein FERRIC REDUCTASE DEFECTIVE 3 (FRD3).¹⁰ With a relatively low specificity, the non-proteinogenic amino acid NA could chelate and transport several essential metal ions in plants. NA promotes the delivery of iron to its target sites and prevents iron precipitation.¹¹ In addition, it helps plant avoid iron toxicity because the chelation inhibits radical production via the Fenton reaction.¹² These essential and protective functions of NA are important for cellular iron distribution in plant tissues. In Arabidopsis, quadruple nas4x-2 mutant, which cannot synthesize any NA, shows strong leaf chlorosis and is sterile. Absence of NA, iron accumulated in the phloem. Sink organs of this mutant were iron deficient, while aged leaves were iron sufficient. NA could also benefit pollen development in anthers and pollen tube passage in the carpels.^13,14

Previous studies revealed that FRD3, a multidrug and toxin efflux protein, facilitates iron chelation to citrate and the subsequent transport of iron-citrate from the root to the shoot.^15-17 The first mutant allele of FRD3 was isolated as a manganese accumulator, hence was first designated man1. man1 was described as a chlorotic and dwarf mutant that contains an increased concentration of iron in the root and displays a constitutive ferric-chelate reductase activity.¹⁸ After FRD3 effluxes citrate into the root vasculature, iron moves through the xylem as a ferric-citrate complex, which is important for the translocation of iron to the shoots. Additionally, the FRD3 citrate effluxer could promote iron nutrition between symplastically disconnected tissues throughout Arabidopsis development. The frd3 fertility defect is mainly caused by pollen abortion and is correlated with the male gametophytic expression of FRD3. The deposits of iron on the surface of aborted pollen grains points to a role for FRD3 and citrate in proper iron nutrition of embryo and pollen.^17,19 Rogers et al. (2002) reported that FRD3 is strongly expressed in Arabidopsis seed and flower, and the frd3 mutant constitutively exhibits iron-deficiency symptoms.²⁰ Consistently, frd3 loss-of-function mutants are defective in early germination and are almost completely sterile, but both defects could be rescued by iron and/or citrate application.¹⁹

Epigenetic Regulation of Iron Homeostasis in Arabidopsis

The term ‘epigenetics’ refers to the changes in gene expression or cellular phenotype that are stably transmitted during mitosis and meiosis without changes in the underlying DNA sequence.²¹ Epigenetic mechanisms play critical roles in the perception of environmental cues by plants, and the regulation of plant biotic and abiotic stress responses.²²

In the last decade, the epigenetic mechanisms involved in iron homeostasis have gradually emerged. For example, Shk1 binding protein 1 (SKB1), which catalyzes the symmetric dimethylation of histone H4R3 (H4R3sme2), is found to participate in iron homeostasis in Arabidopsis. Compared to the wild type, the skb1 mutant exhibited higher iron accumulation in shoots and greater tolerance to iron deficiency. Though the expression of SKB1 was not induced by low iron, the levels of H4R3sme2 mediated by SKB1 were related to the plant iron status. The quantity of SKB1 that associated with chromatin of the 4 bHLH genes (AtbHLH38, AtbHLH39, AtbHLH100 and AtbHLH101) and the levels of H4R3sme2 were higher with increased iron supply and were lower with decreased iron supply. Histone H4R3 dimethylation negatively regulates iron homeostasis by repressing the expression of the 4 bHLH genes and thus iron uptake processes. In other words, iron deficiency may lead to the disassociation of SKB1 from chromatin of the bHLH genes and a decrease of the H4R3sme2 level, which promotes their transcription and enhances the uptake of iron (Fig. 1).²³

Figure 1.

Hypothetical model of the molecular mechanism by which SKB1 regulates iron uptake processes. Under iron-replete conditions, SKB1 binds to the chromatin of bHLH genes and mediates their H4R3sme2 modifications, which could repress bHLH genes transcription (A). Iron deficiency promotes the disassociation of SKB1 from the bHLH genes, which initiates their transcription (B). Dimerization of FIT with other 4 bHLH proteins mediates the transcriptions of IRT1 and FRO2 and finally enhances the capacity of iron uptake (C).

Recently, Xing et al. (2015) reported that histone acetylation also contributes to iron homeostasis in Arabidopsis. The mutation in General control non-repressed protein 5 (GCN5) greatly impaired iron translocation from the root to the shoot. The gcn5 mutant retained much more iron in the root, while the aerial parts of the mutant exhibited phenotypes caused by iron deficiency. High-throughput RNA sequencing together with ChIP assays indicated that 5 genes, including the well-established FRD3, are direct targets of GCN5 in iron homeostasis regulation. Under iron-deficiency conditions, the elevated association of GCN5 to FRD3 locus caused an increase of H3K9/14ac, and finally modulated the dynamic expression of FRD3. However, the iron retention defect in gcn5 was successfully rescued and the fertility was partly restored by overexpressing FRD3. Moreover, iron retention in gcn5 roots was significantly reduced by the exogenous application of citrate, which was consistent with the conclusion that FRD3 was a direct target of GCN5. The downregulation of FRD3, the upregulation of IRT1 and FRO2 and the increased production of root hairs jointly contributed to the accumulation of iron in roots, which might lead to iron toxicity in the root systems of the gcn5 mutant (Fig. 2).²⁴ These 2 studies provide novel insights into the chromatin-based regulation of iron homeostasis in Arabidopsis.

Figure 2.

GCN5 regulates the expression of FRD3 by modulating its H3K9/14ac levels. In gcn5 mutants, the compromised expression of FRD3 leads to the low efficiency of iron translocation from the root to the shoot (➀). The aerial parts of the plant lacks of iron, which might trigger the induction of IRT1 and FRO2 at the root even under iron-replete conditions (➁). In addition, the gcn5 mutant exhibits more and longer root hairs than wild type under normal conditions (➂), which is a common morphology of malnourished plants. These jointly contributed to the accumulation of iron in roots, which might lead to iron toxicity in the root systems of the gcn5 mutant.

Future Perspectives

Genetic and epigenetic manipulations of crops have their own advantages. The relationships between the epigenetic regulation and the nutrition homeostasis have gradually been elucidated, but the application to crop improvement is still out of reach. Because the ‘writers’, ‘readers’ and ‘erasers’ in epigenetics generally regulate the expression of hundreds of genes concurrently and exert pleiotropic functions for plant growth and development, it is impossible to modulate the expression of a single gene or the phenotype of a single trait by simply overexpressing or knocking out the epigenetic regulators. More recently, a newly invented technology, which originated from the research of the RNA-directed DNA methylation (RdDM), has shed light on solving such kind of questions. SUVH2 and SUVH9 act in the downstream of RdDM, and both contain SRA domains capable of binding methylated DNA and functioning to recruit Pol V through DNA methylation.²⁵ Johnson et al. (2014) pointed that tethering SUVH2 with a zinc finger (ZF) to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. They exploited practical application of epigenetic manipulation in Arabidopsis using a ZF to tether SUVH2 to an unmethylated epiallele of FWA, fwa-4. In ZF-SUVH2 transgenic T1 lines, DNA methylation was observed immediately around the ZF binding sites in all cytosine sequence contexts. Importantly, FWA methylation and gene silencing were maintained in T2 plants that had segregated away the ZF-SUVH2 transgene, indicating that targeting by SUVH2 is capable of inducing DNA methylation and gene silencing that can be maintained when the initial trigger is removed.²⁶ These mechanistic findings pave the way to locus-specific chromatin engineering in crops and may help uncover prospects for improving iron nutrient utilization efficiency and crop yields.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work has been executed with financial support from the National Basic Research Program of China (973 Program) (2012CB910900).