Iron Homeostasis In Plants Research Articles

Genome-wide identification and expression analysis of ferric reductase oxidase (FRO) genes in Gossypium spp. reveal their crucial role in iron homeostasis under abiotic and biotic stress

Ferric Reductase Oxidase (FRO) genes are pivotal in iron uptake and homeostasis in plants, yet they are not studied in cotton. Here, we identify and analyze 65 FRO homologs (21 GhFRO, 21 GbFRO, 11 GaFRO, 12 GrFRO) across four Gossypium species (G. hirsutum, G. barbadense, G. arboreum, G. raimondii). FRO exhibit conserved ferric reductase activity and conserved domain structures; Ferric_reduct (PF01794), FAD_binding_8 (PF08022), and NAD_binding_6 (PF08030) across species. Physicochemical properties and subcellular localization analysis provided insights into FRO proteins' functional characteristics, mainly localized to the plasma membrane. Phylogenetic analysis delineates 11 groups, indicating both conserved and divergent evolutionary patterns. Gene structure analysis unveils varying exon-intron compositions. Chromosomal localization shows distribution across A and D genomes, suggesting evolutionary dynamics. Synteny analysis reveals paralogous and orthologous gene pairs subjected to purifying selection. The cis-regulatory elements analysis implicates diverse regulatory mechanisms. Expression profiling highlights dynamic regulation across developmental stages, abiotic and biotic stress conditions. GhFRO interacts with Ca++-dependent protein kinases-10/28-like (CDPKs10/28-like) and metal transporter Natural resistance-associated macrophage protein 6 (Nramp6) to regulate metal ion transport and iron homeostasis. The three-dimensional protein structure prediction suggests potential ligand-binding sites in FRO proteins. Moreover, qRT-PCR analysis of selected eight GhFROs in leaves treated with stress elicitors, MeJA, SA, NaCl, and PEG for 1h, 2h, 4h, and 6h revealed significant downregulation. Overall, this comprehensive study provides insights into FRO gene diversity, evolution, structure, regulation, and function in cotton, with implications for understanding plant iron homeostasis and stress responses.

The transcription factor Dof3.6/OBP3 regulates iron homeostasis in Arabidopsis.

Iron is an essential element for plants. Iron uptake by plants is highly regulated, but the underlying mechanism is poorly understood. Using a truncated fragment of the iron deficiency-responsive bHLH100 gene promoter, we screened the Arabidopsis transcription factor yeast one-hybrid (Y1H) library and identified the DOF family protein, OBP3, as a crucial component of the iron deficiency-signaling pathway. OBP3 is a transcriptional repressor with a C-terminal activation domain. Its expression is induced by iron deficiency. The transgenic lines that overexpress OBP3 exhibited iron overload and premature leaf necrosis, while the obp3 mutant was less tolerant of iron deficiency. It was discovered that OBP3 directly targets the Ib subgroup of bHLH gene promoters. OBP3 interacts with the bHLH transcription factor ILR3 (IAA-LEUCINE RESISTANT3), and their interaction enhances the DNA-binding ability and transcriptional promoting activity of OBP3, resulting in the positive regulation of iron deficiency-response genes. In addition, the E3 Ligase BRUTUS facilitates 26S proteasome-mediated degradation of OBP3 protein to prevent excessive iron uptake in plants. In conclusion, our research emphasizes the vital role of OBP3 in regulating plant iron homeostasis.

CRISPR/Cas9 edited StbHLH47 lines exhibit altered expression profiling of iron regulating genes and increased iron content in Solanum tuberosum

Iron is an essential plant nutrient, and a continuous supply of it is required as it is a key factor in various metabolic processes, including photosynthesis, chlorophyll synthesis, and respiration. Various transcription factors are known to regulate iron homeostasis in plants, and the bHLH transcription factor family is one of them. The StbHLH47 is a homologue of the Arabidopsis POPEYE (PYE), which is known to repress iron homeostasis-related genes in Arabidopsis. Potato is the most consumed vegetable in the world and is low in iron content. We have generated CRISPR/Cas9-edited StbHLH47 lines and performed a detailed analysis of these lines. The analysis revealed that the roots of StbHLH47 edited lines have decreased ferric chelate reductase (FCR) activity compared to the roots of the wild-type (WT) plant. We also observed that CRISPR/Cas9 edited lines have fewer trichomes when compared to the WT plant. The expression of genes associated with iron homeostasis was also measured. Compared to the control, the expression of StbHLH47 was downregulated in the edited lines, while the expression of StNAS4, StOPT3, and StFRO3 was upregulated. This suggests the negative regulation of StbHLH47 in modulating iron. The iron content was also quantified using inductively coupled plasma mass spectrometry (ICP-MS) and found to be increased in the generated transgenic lines when compared to WT plants. Overall, this study reveals that StbHLH47 negatively regulates the expression of iron homeostasis-related genes. StbHLH47 edited lines exhibited decreased FCR activity, changes in phenotype, and increased iron content in the potato plants. Key messageThis study provides novel insight into the role of StbHLH47 in modulating iron content in Solanum tuberosum and controlling the expression of various iron homeostasis-related genes.

GmABCG5, an ATP-binding cassette G transporter gene, is involved in the iron deficiency response in soybean.

Iron deficiency is a major nutritional problem causing iron deficiency chlorosis (IDC) and yield reduction in soybean, one of the most important crops. The ATP-binding cassette G subfamily plays a crucial role in substance transportation in plants. In this study, we cloned the GmABCG5 gene from soybean and verified its role in Fe homeostasis. Analysis showed that GmABCG5 belongs to the ABCG subfamily and is subcellularly localized at the cell membrane. From high to low, GmABCG5 expression was found in the stem, root, and leaf of young soybean seedlings, and the order of expression was flower, pod, seed stem, root, and leaf in mature soybean plants. The GUS assay and qRT-PCR results showed that the GmABCG5 expression was significantly induced by iron deficiency in the leaf. We obtained the GmABCG5 overexpressed and inhibitory expressed soybean hairyroot complexes. Overexpression of GmABCG5 promoted, and inhibition of GmABCG5 retarded the growth of soybean hairy roots, independent of nutrient iron conditions, confirming the growth-promotion function of GmABCG5. Iron deficiency has a negative effect on the growth of soybean complexes, which was more obvious in the GmABCG5 inhibition complexes. The chlorophyll content was increased in the GmABCG5 overexpression complexes and decreased in the GmABCG5 inhibition complexes. Iron deficiency treatment widened the gap in the chlorophyll contents. FCR activity was induced by iron deficiency and showed an extraordinary increase in the GmABCG5 overexpression complexes, accompanied by the greatest Fe accumulation. Antioxidant capacity was enhanced when GmABCG5 was overexpressed and reduced when GmABCG5 was inhibited under iron deficiency. These results showed that the response mechanism to iron deficiency is more actively mobilized in GmABCG5 overexpression seedlings. Our results indicated that GmABCG5 could improve the plant's tolerance to iron deficiency, suggesting that GmABCG5 might have the function of Fe mobilization, redistribution, and/or secretion of Fe substances in plants. The findings provide new insights into the ABCG subfamily genes in the regulation of iron homeostasis in plants.

Genetic basis of the historical iron-accumulating dgl and brz mutants in pea.

The Pisum sativum (pea) mutants degenerate leaves (dgl) and bronze (brz) accumulate large amounts of iron in leaves. First described several decades ago, the two mutants have provided important insights into iron homeostasis in plants but the underlying mutations have remained unknown. Using exome sequencing we identified an in-frame deletion associated with dgl in a BRUTUS homolog. The deletion is absent from wild type and the original parent line. BRUTUS belongs to a small family of E3 ubiquitin ligases acting as negative regulators of iron uptake in plants. The brz mutation was previously mapped to chromosome 4, and superimposing this region to the pea genome sequence uncovered a mutation in OPT3, encoding an oligopeptide transporter with a plant-specific role in metal transport. The causal nature of the mutations was confirmed by additional genetic analyses. Identification of the mutated genes rationalizes many of the previously described phenotypes and provides new insights into shoot-to-root signaling of iron deficiency. Furthermore, the non-lethal mutations in these essential genes suggest new strategies for biofortification of crops with iron.

Exploring the Roles of Arbuscular Mycorrhizal Fungi in Plant–Iron Homeostasis

Arbuscular mycorrhizal fungi (AMF) form a vital symbiotic relationship with plants. Through their extensive hyphal networks, AMF extend the absorptive capacity of plant roots, thereby allowing plants to reach otherwise inaccessible micronutrient sources. Iron, a critical micronutrient involved in photosynthesis and other metabolic processes, often becomes inaccessible owing to its tendency to form insoluble complexes in soil. AMF symbiosis significantly ameliorates this challenge by enhancing iron uptake and homeostasis in plants, altering root architecture, and producing root exudates that improve iron solubility. Moreover, the interaction with diverse soil bacteria, particularly plant growth-promoting rhizobacteria, can potentiate the benefits of AMF symbiosis. Siderophores are low-molecular-weight chelators with iron-binding capacities produced by various microorganisms and plant roots. They play pivotal roles in regulating intracellular iron and have been identified in different mycorrhizal associations, including AMF. While molecular mechanisms behind AMF-mediated iron uptake have been partially explored, the intricate networks involving AMF, plants, siderophores, and other soil microbiota are largely unknown. This review focuses on the multifaceted roles of AMF in plant–iron homeostasis, interactions with soil bacteria, and the potential of siderophores in these processes, emphasizing the possibilities for harnessing these relationships for sustainable agriculture and enhancing plant productivity.

Comprehensive Survey of ChIP-Seq Datasets to Identify Candidate Iron Homeostasis Genes Regulated by Chromatin Modifications.

Vital biochemical reactions including photosynthesis to respiration require iron, which should be tightly regulated. Although increasing evidence reveals the importance of epigenetic regulation in gene expression and signaling, the role of histone modifications and chromatin remodeling in plant iron homeostasis is not well understood. In this study, we surveyed publicly available ChIP-seq datasets of Arabidopsis wild-type and mutants defective in key enzymes of histone modification and chromatin remodeling and compared the deposition of epigenetic marks on loci of genes involved in iron regulation. Based on the analysis, we compiled a comprehensive list of iron homeostasis genes with differential enrichment of various histone modifications. This report will provide a resource for future studies to investigate epigenetic regulatory mechanisms of iron homeostasis in plants.

Photomorphogenic tomato mutants high-pigment 1 and aurea responses to iron deficiency

• There is a close interaction between Fe nutrition and light perception in plants. • The tomato response mechanisms to Fe deficiency is highly light-dependent. • hp-1 tomato mutant is more tolerant to Fe deficiency. • au tomato mutant tend to accumulate less nutrients. Iron (Fe) is a micronutrient for plant development, as constituent of several photosynthesis- and respiration-related proteins and enzymes. Consequently, Fe deficiency leads to chlorosis in leaves and plant growth impairment. It has become increasingly evident that light signals coordinate iron homeostasis in plants. To further address new insights into how light is a fundamental part of Fe deficiency responses, we employed Micro-Tom (wild type, WT) tomato ( Solanum lycopersicum L.) plants and high-pigment 1 ( hp1 ) and aurea ( au ) photomorphogenic mutants, which exhibit an excessive light response and low light perception, respectively. Plant growth, pigment contents, oxidative status, and nutrient profile were analyzed. The results revealed the influence of the different genotypes on Fe deficiency responses. WT and au exhibited plant growth reduction under Fe deficiency. WT, hp1 and au demonstrated that Fe availability and light perception play fundamental roles in chlorophyll and anthocyanin biosynthesis. Lipid peroxidation was not increased for any genotype under Fe deficiency, indicating that mutations in light perception and signaling differentially modulate H 2 O 2 production and scavenging under this condition. Additionally, macronutrients and micronutrients were taken up and distributed differently among the different plant genotypes, tissues and Fe conditions analyzed. In general, the au plants accumulated lower amounts of nutrients (Ca, S, P, Mg, B and Zn) than the WT and hp1 genotypes regardless of the Fe concentrations. Our data clearly indicates that light perception and signaling influence Fe-dependent morphophysiological responses in plants, suggesting possibilities for biotechnological improvement of crops grown under Fe shortage.

Label-Free Quantitative Proteomics in Plant.

Label-free quantitation (LFQ) proteomics, mainly based on the extraction of the peptide (precursor) intensity at the MS1 (mass spectrum 1) level, enables to quantify the relative amount of the proteins among samples. In an LFQ proteomics study, all samples are scanned individually on an advanced mass spectrometer and the chromatographic features of each run are extracted to generate consensus patterns among various runs in the experiment. Here, we describe the LFQ proteomics experimental protocol adapted for plant research, such as plant iron homeostasis.

Minireview: Chromatin-based regulation of iron homeostasis in plants

Plants utilize delicate mechanisms to effectively respond to changes in the availability of nutrients such as iron. The responses to iron status involve controlling gene expression at multiple levels. The regulation of iron deficiency response by a network of transcriptional regulators has been extensively studied and recent research has shed light on post-translational control of iron homeostasis. Although not as considerably investigated, an increasing number of studies suggest that histone modification and DNA methylation play critical roles during iron deficiency and contribute to fine-tuning iron homeostasis in plants. This review will focus on the current understanding of chromatin-based regulation on iron homeostasis in plants highlighting recent studies in Arabidopsis and rice. Understanding iron homeostasis in plants is vital, as it is not only relevant to fundamental biological questions, but also to agriculture, biofortification, and human health. A comprehensive overview of the effect and mechanism of chromatin-based regulation in response to iron status will ultimately provide critical insights in elucidating the complexities of iron homeostasis and contribute to improving iron nutrition in plants.

Emerging roles of protein phosphorylation in plant iron homeostasis.

Remarkable progress has been made in dissecting the molecular mechanisms involved in iron (Fe) homeostasis in plants, especially the identification of key transporter and transcriptional regulatory networks. But how the protein activity of these master players is regulated by Fe status remains underexplored. Recent studies show that major players toggle switch their properties by protein phosphorylation under different Fe conditions and consequently control the signaling cascade and metabolic adjustment. Moreover, Fe deficiency causes changes of multiple kinases and phosphatases. Here, we discuss how these findings highlight the emergence of the protein phosphorylation-dependent regulation for rapid and precise responses to Fe status to attain Fe homeostasis. Further studies will be needed to fully understand the regulation of these intricate networks.

Regulation of plant iron homeostasis by abscisic acid: a review

Iron (Fe) is an important trace element involved in many important plant physiological and metabolic processes such as photosynthesis, respiration and nitrogen metabolism. Plants maintain iron homeostasis through absorption, transporting, storage and redistribution of iron. Iron metabolism is strictly regulated in plants. Iron regulatory transcription factors and iron transporters constitute the regulatory network of plant iron absorption and transport in plants. Ferritin and iron transporter jointly regulate the response to excess iron in plants. In recent years, important progress has been made in understanding how abscisic acid (ABA) regulates iron metabolism in plants. ABA may be used as a signal to regulate the absorption, transportation and reuse of Fe, or to relieve the symptoms of iron stress by regulating the oxidative stress responses in plants. In order to gain deeper insights into the crosstalk of ABA and iron metabolism in plants, this review summarized the mechanisms of iron absorption and transport and metabolic regulatory network in plants, as well as the mechanisms of ABA in regulating iron metabolism. The relationship between ABA and FER-like iron deficiency-induced transcription factor (FIT), iron-regulated transporter 1 (IRT1), and oxidative stress of iron deficiency were highlighted, and future research directions were prospected.

Evidence for a role of nitric oxide in iron homeostasis in plants.

Nitric oxide (NO), once regarded as a poisonous air pollutant, is now understood as a regulatory molecule essential for several biological functions in plants. In this review, we summarize NO generation in different plant organs and cellular compartments, and also discuss the role of NO in iron (Fe) homeostasis, particularly in Fe-deficient plants. Fe is one of the most limiting essential nutrient elements for plants. Plants often exhibit Fe deficiency symptoms despite sufficient tissue Fe concentrations. NO appears to not only up-regulate Fe uptake mechanisms but also makes Fe more bioavailable for metabolic functions. NO forms complexes with Fe, which can then be delivered into target cells/tissues. NO generated in plants can alleviate oxidative stress by regulating antioxidant defense processes, probably by improving functional Fe status and by inducing post-translational modifications in the enzymes/proteins involved in antioxidant defense responses. It is hypothesized that NO acts in cooperation with transcription factors such as bHLHs, FIT, and IRO to regulate the expression of enzymes and proteins essential for Fe homeostasis. However, further investigations are needed to disentangle the interaction of NO with intracellular target molecules that leads to enhanced internal Fe availability in plants.

Iron deficiency triggered transcriptome changes in bread wheat

A series of complex transport, storage and regulation mechanisms control iron metabolism and thereby maintain iron homeostasis in plants. Despite several studies on iron deficiency responses in different plant species, these mechanisms remain unclear in the allohexaploid wheat, which is the most widely cultivated commercial crop. We used RNA sequencing to reveal transcriptomic changes in the wheat flag leaves and roots, when subjected to iron limited conditions. We identified 5969 and 2591 differentially expressed genes (DEGs) in the flag leaves and roots, respectively. Genes involved in the synthesis of iron ligands i.e., nicotianamine (NA) and deoxymugineic acid (DMA) were significantly up-regulated during iron deficiency. In total, 337 and 635 genes encoding transporters exhibited altered expression in roots and flag leaves, respectively. Several genes related to MAJOR FACILITATOR SUPERFAMILY (MFS), ATP-BINDING CASSETTE (ABC) transporter superfamily, NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family and OLIGOPEPTIDE TRANSPORTER (OPT) family were regulated, indicating their important roles in combating iron deficiency stress. Among the regulatory factors, the genes encoding for transcription factors of BASIC HELIX-LOOP-HELIX (bHLH) family were highly up-regulated in both roots and the flag leaves. The jasmonate biosynthesis pathway was significantly altered but with notable expression differences between roots and flag leaves. Homoeologs expression and induction bias analysis revealed subgenome specific differential expression. Our findings provide an integrated overview on regulated molecular processes in response to iron deficiency stress in wheat. This information could potentially serve as a guideline for breeding iron deficiency stress tolerant crops as well as for designing appropriate wheat iron biofortification strategies.

Connecting the negatives and positives of plant iron homeostasis.

This article is a Commentary on Tissot et al., 223: 1433–1446

PRC2-Mediated H3K27me3 Contributes to Transcriptional Regulation of FIT-Dependent Iron Deficiency Response.

Iron is an essential micronutrient for nearly all organisms, but excessive iron can lead to the formation of cytotoxic reactive oxygen species. Therefore, iron acquisition and homeostasis must be tightly regulated. Plants have evolved complex mechanisms to optimize their use of iron, which is one of the most limiting nutrients in the soil. In particular, transcriptional regulation is vital for regulating iron in plants, and much work has revealed the role of transcription factors on this front. Our study adds novel insights to the transcriptional regulation of iron homeostasis in plants by showing that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency. We provide evidence that FIT-dependent iron acquisition genes, IRT1 and FRO2, as well as FIT itself are direct targets of PRC2-mediated H3K27me3. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, induction of FIT, FRO2, IRT1, and other FIT-regulated genes in roots is significantly higher under iron deficient conditions than in wild type. Furthermore, we observe that clf mutants are more tolerant to iron deficiency than wild type, indicating that gene expression levels appear to be limiting the plants ability to access iron. We propose that H3K27me3 attenuates the induction of FIT-target genes under iron deficiency and hypothesize that this may serve as a mechanism to restrict the maximum level of induction of iron acquisition genes to prevent iron overload.

FIT (Fer-like iron deficiency-induced transcription factor) in plant iron homeostasis: genome-wide identification and bioinformatics analyses

FIT (Fer-like iron deficiency-induced transcription factor) is one of the key regulators in uptake of iron (Fe) for plants. In this study, FIT genes from 10 plant species were identified at genome-wide scale and analyzed by computational tools. Our analyses showed that subcellular localizations of all FITs were in nucleus and FITs were generally acidic proteins. While a clear monocot–dicot divergence was observed, exon numbers were found as three and four for monocots and dicots, respectively. Arg and Leu were established as most repetitive amino acid residues in conserved regions of all FITs. All FITs had different shape and pocket numbers along with different secondary and tertiary structures despite they contained same domain structure as PF00010 (helix-loop-helix DNA-binding domain). We observed that FIT is an important target of several biological processes and a component of various metabolic pathways regulating root development and stress responses on exposure to various environmental stressors. Moreover, FITs seem to be located in heterochromatic area of chromosome and its transcription may also be upregulated on the exposure of stressors. The FITs were established to interact with Pi and Cu uptake machinery, other than iron acquisition, under Pi and Cu deficiency. Consequently, FITs can be proposed to take part in different and specific metabolic pathways together with Fe acquisition. The miRNA analyses showed that miRNAs targeting FITs take part in abiotic and biotic stress metabolism together with Pi uptake mechanism. Lastly, involvement of FIT in abiotic stress response was stimulated by phytohormones such as ABA and ethylene.

Hemerythrin E3 Ubiquitin Ligases as Negative Regulators of Iron Homeostasis in Plants.

Iron (Fe) is an essential nutrient for plants, but at the same time its redox properties can make it a dangerous toxin inside living cells. Homeostasis between uptake, use and storage of Fe must be maintained at all times. A small family of unique hemerythrin E3 ubiquitin ligases found in green algae and plants play an important role in avoiding toxic Fe overload, acting as negative regulators of Fe homeostasis. Protein interaction data showed that they target specific transcription factors for degradation by the 26S proteasome. It is thought that the activity of the E3 ubiquitin ligases is controlled by Fe binding to the N-terminal hemerythrin motifs. Here, we discuss what we have learned so far from studies on the HRZ (Hemerythrin RING Zinc finger) proteins in rice, the homologous BTS (BRUTUS) and root-specific BTSL (BRUTUS-LIKE) in Arabidopsis. A mechanistic model is proposed to help focus future research questions towards a full understanding of the regulatory role of these proteins in Fe homeostasis in plants.

Crystal structure of plant vacuolar iron transporter VIT1.

The iron ion is an essential cofactor in several vital enzymatic reactions, such as DNA replication, oxygen transport, and respiratory and photosynthetic electron transfer chains, but its excess accumulation induces oxidative stress in cells. Vacuolar iron transporter 1 (VIT1) is important for iron homeostasis in plants, by transporting cytoplasmic ferrous ions into vacuoles. Modification of the VIT1 gene leads to increased iron content in crops, which could be used for the treatment of human iron deficiency diseases. Furthermore, a VIT1 from the malaria-causing parasite Plasmodium is considered as a potential drug target for malaria. Here we report the crystal structure of VIT1 from rose gum Eucalyptus grandis, which probably functions as a H+-dependent antiporter for Fe2+ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five membrane-spanning domains, with an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and connects to a three-helical bundle, triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in their transport. These mechanistic insights will provide useful information for the further design of genetically modified crops and the development of anti-malaria drugs.

The Transcriptional Control of Iron Homeostasis in Plants: A Tale of bHLH Transcription Factors?

Iron is one of the most important micronutrients in plants as it is involved in many cellular functions (e.g., photosynthesis and respiration). Any defect in iron availability will affect plant growth and development as well as crop yield and plant product quality. Thus, iron homeostasis must be tightly controlled in order to ensure optimal absorption of this mineral element. Understanding mechanisms governing iron homeostasis in plants has been the focus of several studies during the past 10 years. These studies have greatly improved our understanding of the mechanisms involved, revealing a sophisticated iron-dependent transcriptional regulatory network. Strikingly, these studies have also highlighted that this regulatory web relies on the activity of numerous transcriptional regulators that belong to the same group of transcription factors (TF), the bHLH (basic helix-loop-helix) family. This is best exemplified in Arabidopsis where, to date, 16 bHLH TF have been characterized as involved in this process and acting in a complex regulatory cascade. Interestingly, among these bHLH TF some form specific clades, indicating that peculiar function dedicated to the maintenance of iron homeostasis, have emerged during the course of the evolution of the green lineage. Within this mini review, we present new insights on the control of iron homeostasis and the involvement of bHLH TF in this metabolic process.