# Untangling iron threads: a deep dive into plant intracellular pools

Alexandra Leskova, Tou Cheu Xiong, Stéphane Mari, Catherine Curie

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Alexandra Leskova, Tou Cheu Xiong, Stéphane Mari, Catherine Curie. Untangling iron threads: a deep dive into plant intracellular pools. Quantitative Plant Biology, In press, 6 (e14), 10.1017/qqb.2025.11.1. hal-04901799

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# Untangling iron threads: A deep dive into plant intracellular pools

Alexandra Leskova, Tou C. Xiong, Stéphane Mari and Catherine Curie*

IPSiM, University of Montpellier, CNRS, INRAE, Institut Agro, Montpellier, France

## Review

**Cite this article:** A. Leskova et al. Untangling iron threads: A deep dive into plant intracellular pools. *Quantitative Plant Biology*, 6:e14, 1–8
https://dx.doi.org/10.1017/qpb.2025.11

Received: 1 November 2024

Revised: 2 March 2025

Accepted: 3 March 2025

**Keywords:**
inter-organelle flux; ion quantification; iron pool; metal sensor; single cell analysis.

**Corresponding author:**
Catherine Curie;
Email: catherine.curie@cnrs.fr

A.L. and T.C.X. co-first authors.

**Associate Editor:**
Ingo Dreyer

## Abstract

Iron (Fe) is an essential element in plants, involved in numerous metabolic processes including photosynthesis. Its cellular concentration must be regulated accurately to avoid toxicity while meeting metabolic demands. This review explores the distribution, dynamics, and regulation of Fe pools in plant cells, focusing on recent advances in imaging and quantification techniques. We discuss the major Fe compartments—chloroplasts, vacuoles, apoplasts—and their interaction to maintain Fe homeostasis, as well as novel methodologies like single-cell ICP-MS that have transformed our understanding of Fe localization. By summarizing the current knowledge on intracellular Fe dynamics and the complex interplay between different Fe pools, we provide insights into the mechanisms that underpin Fe regulation in plants, which is crucial for future breeding programs aimed at improving plant resilience and nutritional quality.

## 1. Introduction

As a cofactor of numerous enzymes and a component of redox chains, iron (Fe) is indispensable in all forms of life, playing a role in many essential metabolic processes including photosynthesis in plants. Its concentration in plant cells must be tightly controlled to support metabolic needs without inducing toxicity. Regulating Fe concentration is challenging as plants must adapt to variations of Fe availability in soils. Furthermore, environmental cues such as drought or elevated CO₂ also impact Fe content in plants. The adaptive response to Fe-deficiency is decoded and orchestrated by a well characterized cascade of transcription factors that are reviewed elsewhere (Vélez-Bermúdez & Schmidt, 2023). In addition to the regulation of root uptake, identifying and quantifying Fe pools and their dynamics in plant cells is essential to understand the mechanisms of Fe homeostasis and a prerequisite to adapt breeding programs.

As Fe-containing proteins participate in most metabolic reactions, Fe is found in all tissues and cell compartments. Cutting-edge imaging techniques have allowed a lip forward in the description of the main Fe pools in plant cells (Kim et al., 2006; Roschztardtz et al., 2009). Depending on the tissue, Fe is observed in high amounts in chloroplasts, vacuole, cell wall and nucleolus (Roschztardtz et al., 2013; Roschztardtz, Grillet et al., 2011; Roschztardtz, Séguéla-Arnaud, et al., 2011).

The Fe pools are complex and are comprised of (i) the static pool, which is made of Fe tightly bound to proteins, e.g. in ferritins that sequester Fe in the chloroplasts, or to organic ligands such as Fe-phytate complexes in the vacuole; (ii) The labile pool, chemically uncharacterized yet, which is composed of loosely bound, redox-active and readily available Fe for metabolic reactions (i.e. Fe-citrate, Fe-nicotianamine, Fe-peptides...) (Koppenol & Hider, 2019). Labile Fe must be carefully regulated because of its potential to generate reactive oxygen species. But there is more than meets the eye here since Fe also exists under two redox forms, Fe²⁺ and Fe³⁺, that differ in their biological functions. Thus, techniques devoted to detect and measure Fe in cells must be carefully selected based on the type of iron to be measured.

Traditional tools to study intracellular Fe content rely primarily on ICP-MS-based measurements of bulk samples, which inform on Fe content at the tissue scale. More recently, the advent of dedicated and selective imaging tools such as energy dispersive X-ray analyses (EDX) (Lanquar et al., 2005), synchrotron radiation X-Ray Fluorescence (XRF) (Kim et al., 2006), or the Perls-DAB histochemical staining method (Roschztardtz et al., 2009) have allowed

© The Author(s), 2025. Published by Cambridge University Press in association with John Innes Centre. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (https://creativecommons.org/licenses/by/4.0.), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited.

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precise mapping of Fe at the cell or even subcellular levels, however they fail to give an accurate value of Fe concentration. Recently, single cell approaches have allowed quantifying Fe in isolated cells.

This review attempts to summarize the current knowledge of the major, observable, plant intracellular Fe pools, their distribution and function in the main organelles, with a special emphasis on the methods used to achieve their quantification, and further describes the understanding that emerges on the dynamic interplay between Fe pools of different cellular compartments to attain the set point of Fe concentration in a cell.

## 2. Cell-specific and single cell Fe analyses

Elemental measurement through ICP-MS has been very recently adapted to single cells and revolutionized the quantification of symplastic elements. Two studies report their utilization to determine the ionomic content of cells from different root tissue layers or pollen grains (Giehl et al., 2023; Jiménez-Lamana et al., 2023).

The first study employed cell type-specific ICP-MS, following cell sorting based on fluorescence of tissue-specific reporter genes and established the ionomic profile across Arabidopsis root tissue layers (Giehl et al., 2023). This study revealed the existence of a radial Fe gradient within the primary root, with cellular Fe concentrations increasing from epidermis to endodermis

(Figure 1a). Furthermore, challenging this multi-elemental analysis by exposing plants to various nutritional stresses allowed monitoring nutrient-specific accumulation responses, which follow precise distribution patterns. For example, manganese (Mn) abundance increased in trichoblasts in response to Fe deficiency. The specific Mn retention in this cell-type is a consequence of active loading of Mn into the vacuoles, which protects against Mn overaccumulation in the shoots, emphasizing the role of this cell-type in the complex interplay between Fe and Mn. A specificity of single cell approaches of complex plant tissues is that they require lysis of the cells for sorting, which excludes cell wall-bound fractions. In contrast to radial gradients of symplastic Fe revealed by cell-specific ICP-MS, the majority of labile and total Fe in Arabidopsis roots, shown respectively by Fe imaging using synthetic probes and laser ablation coupled to ICP-MS (LA-ICP-MS), accumulate in the outer cell layers (Alcon et al., 2024; Persson et al., 2016). There, most of labile Fe species accumulate in the apoplastic spaces (Alcon et al., 2024; Roschzttardtz et al., 2013) as discussed below. Together, these independent studies highlight the need for complementary methodologies to evaluate the distribution of the different Fe species and pools.

In contrast to the above-described cell-type ionomic analysis that quantified average Fe concentration from a population of sorted cells, a second study achieved absolute quantification of metal ions in individual and intact cells, by applying single

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Quantitative representation of Fe distribution across different cell layers in the primary root of *Arabidopsis*. (a) Schematic representation of the symplastic Fe concentration measured via ICP-MS in tissue-specific cell-sorted root protoplasts, based on data from Giehl et al. (2023). (b) Distribution of labile $Fe^{2+}$ in the Arabidopsis root showing polar localization in the epidermis (based on data from Alcon et al., 2024). Left: Longitudinal confocal laser scanning microscopy images of the differentiation and mature zones of the primary root of Arabidopsis stained with an $Fe^{2+}$-specific fluorescent probe and counter-stained with propidium iodide. The pictures show enrichment of $Fe^{2+}$ in cell walls and opposite polarity in the two root stages. Right: Schematic representation of the distribution and relative quantification of labile $Fe^{2+}$ pools in the same two root regions. Fe levels are represented using a color-coded scale. Magenta LUT: SiRhoNox-1, $Fe^{2+}$; Blue LUT: Propidium iodide, PI. Ep = epidermis; c = cortex; en = endodermis. Scale bar = 20 µm.</figcaption></figure>
cell ICP-MS (SC-ICP-MS) to the pollen grain of Arabidopsis (Jiménez-Lamana et al., 2023). Similarly to unicellular organisms, dehiscent pollen grains can be sorted prior to ICP-MS quantification without the need for cell wall lysis, and are therefore easily amenable to elemental quantification at the whole cell level (Jiménez-Lamana et al., 2023; Miyashita et al., 2014; Shen et al., 2019). SC-ICP-MS of pollen grains detected Fe and Mn in the femtogram range (≈6 fg Fe and 7 fg Mn). For comparison, one epidermis protoplast with similar diameter (≈20 µm) contains in average 18 fg of Fe and 5 fg of Mn (Giehl et al., 2023). The study also revealed that a soluble fraction of Fe, but not Mn, is loosely bound to the cell wall and readily released in the medium upon resuspension in water (Jiménez-Lamana et al., 2023). This suggests that in pollen, like in epidermal cells as discussed below, Fe is more likely to accumulate in a labile form at the cell wall, raising the question of the exact nature and physiological role of this Fe pool.

### 3. Chloroplasts as gatekeepers of intracellular Fe balance

Fractionation of leaf cells established that the majority of Fe in plant shoot is localized within chloroplasts, representing approx. 70% of the total leaf tissue content (Shikanai et al., 2003). There, Fe is used in photosynthesis, each electron transport chain containing 22 Fe atoms, as well as for the biosynthesis of heme and Fe-S clusters (Kroh & Pilon, 2020). When Fe supply is abundant or upon senescence, it is sequestered by the storage protein Ferritin within the stroma. Defect of storage increases the pool of free Fe, which has the potential to catalyze reactive oxygen species (ROS) production through the Fenton reaction (Briat et al., 2010). Aberrant ROS are also generated by Fe deficiency as a result of disturbed photosynthesis and impaired antioxidant capacity of the chloroplastic Fe-containing Superoxide Dismutase (FeSOD) (López-Millán et al., 2016). Controlling Fe content in chloroplasts is therefore of utmost importance. The transporters that help Fe cross the chloroplast envelope are not very well defined. Fe presumably enters the chloroplast by the PIC1 permease *via* its interaction with NiCo (Duy et al., 2007, 2011), although several pieces of data point to a more general role in nutrient or even protein import (Eitinger et al., 2005; Teng et al., 2006), and necessitates a prior reduction mediated by the FRO7 ferric reductase at its surface (Jeong et al., 2008). At specific developmental stages such as cotyledons or senescent leaves, Fe is exported from the chloroplast by the YSL4 and YSL6 transporters (Divol et al., 2013). To date, this couple of transporters still lacks an identified substrate, since the presence of the usual substrate of YSL family members, the metal binding-ligand nicotianamine, has not yet been measured in chloroplasts. YSL4 and YSL6 being members of the large family of oligopeptide transporters, their substrate is predicted to be a small peptide with Fe-binding capacities, the nature of which remains to be identified in chloroplasts. With FPN3, which is produced both in mitochondria and chloroplasts, indirect evidences suggest a role in Fe efflux from the two organelles (Kim et al., 2021), but this conclusion would be greatly strengthened by imaging the modification of the organellar Fe pools in the knockout mutant.

In agreement with Fe measurements in isolated chloroplasts, histochemical staining of Fe by the user-friendly Perls-DAB method, which enables to visualize and map static Fe³⁺ in fixed tissues, has established that in Arabidopsis leaves, the strongest signal is indeed observed in the chloroplasts, part of it as dot-like structures that were identified as Fe stably bound to Ferritins (Roschztardtz et al., 2013). Chloroplastic Fe is particularly impacted by insufficient Fe supply. Cellular fractionation and µXRF

of cucumber leaves showed that plants grown under Fe deficiency accumulate undetectable amounts of Fe in chloroplasts (Vigani et al., 2018). Nevertheless, accurate quantification of Fe in a single chloroplast and in response to stresses has not been reported. Furthermore, the full inventory of Fe species in the stroma has not been established. Besides Fe³⁺-Ferritin, which is easy to visualize using histochemical staining of Fe, several labile forms of Fe could co-exist in chloroplasts, including Iron-Sulfur (Fe-S) clusters, the assembly of which takes place for a part in the chloroplast, and labile species with ligands that are knowingly present in chloroplasts such as Fe³⁺-citrate, Fe³⁺-malate and Fe²⁺-ascorbate.

### 4. Iron cycling and storage in seed vacuoles

The transport and storage of Fe in vacuoles of seed tissues is, to date, the best documented example of the dynamic cycling of iron in and out of an organelle. Two decades ago, EDX on Arabidopsis seeds provided the first quantitative characterizations of the mineral content of globoïds, which are phytate-rich intra-vacuolar structures known to accumulate high concentrations of phosphate and minerals such as K, Mg, Ca and Fe (Lanquar et al., 2005; Lott et al., 2001). Elemental imaging approaches such as micro Particle-Induced X-ray Emission (µPIXE) and µXRF showed that the globoïds of the aleurone layer of wheat grains are a hot spot for Fe and phosphorus (P), both associated in Fe-phytate complexes, as established by X-ray Absorption Near Edge Structure (XANES) (De Brier et al., 2016; Singh et al., 2014).

Interestingly, Fe isotope uptake coupled with nanoscale-secondary ion mass spectrometry (NanoSIMS) in wheat grains revealed Fe enrichment in endospermic vesicles of different sizes, smaller vesicles devoid of-, and large vesicles bound with phosphorus-oxygen (PO) ligand, likely phytate (Sheraz et al., 2021). The authors were able to capture fusion of new ⁵⁷Fe-enriched vesicles with PO-bound vesicles containing only natural (older) ⁵⁶Fe isotope. This raises the question whether these Fe-rich vesicles of different composition are eventually shuttled to the vacuoles and/or fuse with the plasma membrane to participate in cell-to cell transport.

Although the speciation of Fe in Arabidopsis is similar to wheat, µXRF mapping, as well as histochemical staining of Fe with Perls/DAB, have independently shown that Fe is stored in endodermal cells, a specific cell layer that surrounds the provascular bundles of the embryo (Kim et al., 2006; Roschztardtz et al., 2009). The VIT1 protein is responsible for the transport of Fe²⁺ in the vacuoles of these cells and this Fe pool builds up all along embryo development as Fe³⁺-phytate (Lanquar et al., 2005; Roschztardtz et al., 2009). Preventing the upbuilding of this specific vacuolar storage in a *vit1* mutant provokes mislocalization of Fe atoms in vacuoles of the sub-epidermal cells that otherwise store Mn, through the transport activity of the Mn/Fe transporter MTP8 (Eroglu et al., 2017). During germination, Fe ions are retrieved from the vacuoles through the efflux activity of the ferrous iron transporters NRAMP3 and NRAMP4 (Lanquar et al., 2005). Inactivation of *NRAMP3* and *NRAMP4* impaired Fe remobilization and severely compromised growth of the seedlings in Fe-limiting environments (Lanquar et al., 2005; Roschztardtz et al., 2009). Interestingly, the mis-localization of Fe in the *vit1* mutant also impaired its remobilization, suggesting that, in this case, the capacity to retrieve Fe from sub-epidermal vacuoles is absent (Kim et al., 2006). The fact that iron is transported in and out of the vacuoles as Fe²⁺ but stored as Fe³⁺ implies that a redox
regulation must occur, to ensure proper cycling of Fe atoms in this compartment. The Multidrug And Toxic compound Efflux (MATE) protein AtDTX25 was recently identified as a component of this Fe redox regulation. This membrane protein mediates the transport of ascorbate, a potent Fe³⁺-reducing compound, in vacuoles, and its role is crucial during germination to mediate the reduction of phytate-bound Fe³⁺ and therefore generate Fe²⁺, the substrate of the efflux transporters NRAMP3 and NRAMP4 (Hoang et al., 2021).

## 5. Apoplast as an important buffer compartment of Fe

Apoplastic iron has long been recognized as an important Fe reservoir in plants. In roots of several plant species, apoplastic Fe was shown to constitute approx. 40 to 90% of the total Fe (Bienfait et al., 1985; Giehl et al., 2023; Strasser et al., 1999). Thus, apoplastic Fe is established as one of the major Fe pools in plants. Its size changes dynamically in response not only to Fe availability but also to other nutrients stresses (NH₄, P) and to pathogen attack (Liu et al., 2023; Xing et al., 2021). Perls-DAB staining of Fe³⁺ revealed abnormal Fe deposition in the apoplastic compartment of Arabidopsis mutants with impaired Fe transport, such as *irt1* *nramp1*, *frd3*, and *ferritin* (*fer134*) (Castaings et al., 2016; Roschztardtz et al., 2013; Roschztardtz, Séguéla-Arnaud, et al., 2011). Though useful for comparative analysis, this method does not provide quantitative measurements of Fe concentrations.

An alternative method consists in the use of fluorescent probes that enable the visualization of metals *in vivo* and offer a more quantitative approach (Alcon et al., 2024; Maniero et al., 2024; Platre et al., 2022). Unlike the Perls-DAB method, recently developed fluorescent Fe probes, applied in combination, allow to simultaneously and distinctively detect the labile Fe redox forms Fe²⁺ and Fe³⁺ (Alcon et al., 2024). Utilization of these probes to investigate the distribution of the labile Fe pools in Arabidopsis root tissues indicated that cell walls, in particular at the periphery of epidermal cells, contains large amounts of labile Fe. Furthermore this study showed that the redox status of Fe is tightly regulated as the root ages since root apex is enriched in Fe³⁺, whereas the elongation and differentiation zones show a complex pattern of both Fe²⁺ and Fe³⁺. In the epidermis, Fe²⁺ ions precisely mapped to the plasma membrane and apoplastic space. In contrast, Fe³⁺ was rather enriched inside the cell, in a large compartment that resembles the central vacuole.

Remarkably, the method from Alcon et al., (2024) uncovered polar distribution of Fe²⁺ ions that are exclusively located at the outer side of the epidermal cells (Figure 1b). Incidentally, the plasma membrane bound-, Fe deficiency-induced, Ferric reductase FRO2 harbours the same polar pattern (Martín-Barranco et al., 2020). This is consistent with the finding of Alcon et al., (2024) that this epidermal Fe²⁺ pool is partially lost in the Fe-deprived *fro2* mutant. Interestingly, the Fe²⁺ polar pattern observed in the differentiation zone switches side in the mature root zone where Fe²⁺ ions accumulate in the inner side of the epidermal cell, i.e. facing the cortex (Figure 1b).

Our knowledge of the role of different cell wall-bound Fe forms is rudimentary. A recent study in Arabidopsis revealed that Fe deficiency triggers galactosylation of the cell wall polymer rhamnogalacturonan-II (RG-II) by inducing the expression of the glycosyltransferase-encoding *Cdi* gene. This, in turn, imposes cell wall modifications that promote desorption of Fe atoms bound to the cell wall and increases tolerance of the plants to Fe deficiency (Peng et al., 2021). Thus upon Fe limiting conditions, Fe can be

re-assimilated from apoplastic pools. Nevertheless, labile apoplastic Fe, particularly Fe³⁺, can negatively impact the growth as observed in phosphate (Pi) deficient conditions. In this situation, Fe³⁺ accumulates in the apoplastic spaces of root apical meristem, owing to the activity of the plasma membrane-localized ferroxidase LPR1. There, iron accumulation induces the efflux of malate, through ALMT1, leading to the formation of Fe³⁺-malate complex in the apoplast. This results in the accumulation of ROS that in turn trigger callose deposition and stiffening of the cell walls, slowing down elongation (Müller et al., 2015). To equilibrate the Fe²⁺/Fe³⁺ ratio in the root tip apoplast, plants have evolved a Fe³⁺ reduction strategy through the production of an ascorbate-dependent ferric reductase related to the CYBDOM family (Clúa et al., 2024; Maniero et al., 2024). Biotic stress conditions, such as pathogen attack can also contribute either to withholding or on the contrary to boosting local apoplastic pools (reviewed in Liu et al., 2023). Thus, apoplastic Fe pools can be either beneficial or detrimental to plant tolerance depending on the form of Fe, type of stress and particular root zone or cell.

## 6. Dynamic interplay between organellar Fe pools

Adequate distribution of Fe between cell compartments ensures plant growth and development under fluctuating Fe conditions. To safeguard such balance when plants face adverse conditions, dynamic fluxes of Fe occur between the different organelles. In Arabidopsis, these fluxes are best illustrated by the communication between vacuolar Fe store and chloroplastic Fe pool since their respective size impacts each other's. When Fe efflux from the chloroplast was shut off as a consequence of inactivating the *YSL4* and *YSL6* genes, vacuolar Fe remobilization by NRAMP3 and NRAMP4 was inhibited (Divol et al., 2013). Conversely, enhancing chloroplastic Fe efflux *via* the overexpression of *YSL4* and *YSL6* led to reduced Fe concentration in the vacuole. Likewise, enlarging the vacuolar Fe pool via either inactivating *NRAMP3* and *NRAMP4* or over-expressing *VIT1*, reduced accumulation of Ferritins, the abundance of which being a readout of the chloroplast Fe status (Ravet et al., 2009).

A similar dialog apparently exists between intracellular and apoplastic Fe pools. Ferritin-less leaves of the Arabidopsis *fer1/3/4* triple mutant accumulate dramatic amounts of Fe in the apoplast of mesophyll cells (Roschztardtz et al., 2013). It is not clear whether this protective response to the lack of Fe buffering system in the chloroplast results from inhibition of Fe uptake from the intercellular space or from stimulation of a still elusive Fe efflux activity at the plasma membrane. The MCO1 and MCO3 apoplastic ferroxidases, provide another striking example of this dialog. Inactivating *MCO1* and *MCO3* genes, which are highly induced in Fe excess conditions, provoked an overaccumulation of Fe-Ferritin complexes in the chloroplasts, illustrating that the strict control of the Fe redox status is crucial to regulate its uptake from the apoplastic compartment (Brun et al., 2022). Taken together, these findings stress the role of the nature of the intracellular Fe pools and the ongoing communication between them to optimize metabolic functions and plant growth.

## 7. Concluding remarks and open questions

Methodological advances have enabled to map major pools of both tightly bound Fe and labile redox Fe forms within plant cells. Table 1 summarizes the strengths, limitations and the
<table><caption>**Table 1.** Comparison of advanced analytical methods for measuring iron content and dynamics in plant tissues and cells</caption><thead><tr><th></th><th>Selectivity</th><th>Measurement detection limit</th><th>Observed spatial<br>resolution limit (examples)</th><th>Sample preparation<br>Destructive/live</th><th>Accessibility cost</th><th>References</th></tr></thead><tbody><tr><td>Perls-DAB</td><td>Static Fe<sup>3+</sup></td><td>Qualitative</td><td>Tissular to <b>Subcellular</b> (nucleus, nucleolus, vacuoles, globoids, Ferritins, cell wall)</td><td>Chemical fixation<br>Destructive</td><td><b>Accessible inexpensive</b></td><td>Roschztardtz et al. (2013)</td></tr><tr><td>SC-ICP-MS</td><td>Total iron</td><td><b>Absolute &lt; fg</b></td><td><b>Single cell</b> (pollen grains, root protoplasts)</td><td>Isolated cells<br>Destructive</td><td>Accessible expensive</td><td>Jiménez-Lamana et al. (2023), Giehl et al. (2023)</td></tr><tr><td>Fe redox probes<br>SiRhoNox-1<br>MPNBD</td><td>Labile Fe<sup>2+</sup><br>Labile Fe<sup>3+</sup></td><td>Semi-quantitative<br>and <b>dynamic</b><br>&lt;1 µM (Fe<sup>2+</sup>)<br>&lt;70 µM (Fe<sup>3+</sup>)</td><td>Tissular to <b>Subcellular</b> (cell wall, vacuole, vesicles)</td><td><b>No preparation</b><br><b>Live tissue</b></td><td><b>Accessible inexpensive</b></td><td>Alcon et al., 2024</td></tr><tr><td>nXRF, µXRF</td><td>Total iron</td><td>Semi-quantitative &lt; ppm</td><td>Tissular to <b>Subcellular</b> (vacuole, nucleus, nucleolus, plastids, cell wall)</td><td><b>Environmental or<br>cryopreparation</b><br>Destructive</td><td>Limited accessibility pending proposal<br>acceptance by Synchrotrons</td><td>Kim et al. (2006),<br>Roschztardtz et al. (2011),<br>Punshon et al. (2012)</td></tr><tr><td>Nano-SIMS</td><td>Total iron</td><td>Semi-quantitative &lt; ppm</td><td><b>Subcellular</b> (vacuoles, globoids, vesicles)</td><td>Lengthy preparation<br>Destructive</td><td>Limited accessibility expensive</td><td>Sheraz et al. (2021)</td></tr><tr><td>LA-ICP-MS</td><td>Total iron</td><td>Semi-quantitative<br>&lt;10 ppm</td><td><b>Cell-type</b> (root)</td><td><b>Freeze-dry</b><br>Destructive</td><td>Limited accessibility expensive</td><td>Persson et al. (2016)</td></tr></tbody></table>

Note: Strengths of each method are indicated in bold.
complementary aspects of these methodologies. However, the identity of the Fe pools in smaller organelles like mitochondria, Golgi apparatus, lyzosomes, or peroxisomes, which all contain important Fe-dependent enzymes, has not yet been deciphered. Visualization and quantification of these pools, an endeavour given how petite these organelles are, could hypothetically be achieved by organellar immunoprecipitation using antibodies targeting protein tags residential to the organelle or organellar subdomains of interest (Drakakaki et al., 2012). With ever-increasing sensitivity of the analytical methods, Fe content in these fractions could be resolved in the future.

Astonishing enrichment of Fe has also been detected in the nucleus and nucleolus of several plant tissues (Hilo et al., 2017; Ideas et al., 2017; Roschztardtz, Grillet, et al., 2011), the function of which is obscure. (Montacié et al., 2023) found aberrant ribosomal RNA synthesis in the Fe-deficient nicotianamine triple mutant nas124. But it is yet unclear in which form, free or associated to a nucle(ol)ar protein, Fe is present in this compartment.

The wealth of transporters that take in charge the movement of Fe between cell compartments is starting to unveil, yet many of the important ones are still missing from the inventory. Notably, it remains to be established whether Fe efflux activity at the cell surface or vesicular secretion of Fe or both take part in maintaining intracellular Fe concentration. Evidence in favour of the existence of such cellular Fe export mechanisms in plants is still lacking. It is possible that there is sufficient buffering capacity for excess Fe, via storage in vacuoles or retention in the apoplasts, to render cellular Fe efflux activity dispensable. The interplay that exists between Fe pools likely involves regulation of intracellular transporter genes, either already known or as yet uncharacterized, as well as a complex set of signals for inter-organellar communication, but this elaborate network has not begun to be explored.

The speciation of Fe is intricately linked with its redox status, since the ligands have very different affinities towards Fe²⁺ and Fe³⁺ (i.e. the stability constant for Fe³⁺-citrate is 8 orders of magnitude higher than that of Fe²⁺-citrate). Therefore, ferric reduction and fer- roxidation activities play a central role in the regulation of Fe spe- ciation that will, in turn, affect its mobility and reactivity in the cell. Compared to ferric reduction systems (FRO, ascorbate, ascorbate- dependent CYB-DOM proteins), ferroxidases have received much less attention, despite the fact that they might be as important for Fe distribution in cells. The recent reports on the ferroxidases LPR1 (Naumann et al., 2022; Xu et al., 2022) and MCO1 and MCO3 may likely be the tip of the iceberg in this rather unexplored domain.

There is still a long journey ahead before we can better quantify and localize the numerous Fe pools that are present within a plant cell. However, milestones have been set recently, for example with single cell studies or with selective probes to image the redox forms of Fe. The advent of single cell methods and the newly implemented cell type-specific ICP-MS techniques now offer a robust platform for high-throughput quantitative analysis of cel- lular metal fingerprints across various tissues, growth conditions and genetic backgrounds. The utilization of fluorescent probes to measure Fe concentrations in plant cells is not flawless. Obstacles that persist include the difficulty to control probe concentration within the cell, or the fact that pH variations that exist between cell compartments or upon stress affect measurement accuracy. Nevertheless, the probes represent an invaluable tool for in the future undertaking the immense task of deciphering Fe content and redox status in all tissues, cells and subcellular compartments. Applications are numerous, including (i) tackling the substrate and activity of candidate transporters, (ii) addressing the dynamics

of Fe by tracking probe fluorescence in living cells. Efforts now need to focus on the development of genetic sensors, which are more efficient than chemical probes to visualize dynamic fluxes of all forms of labile Fe in live tissues and will thus help decipher the hierarchy of Fe shuffling between intracellular pools and the cellular signaling pathways at play.

**Data and coding availability statement.** No data or code were generated as part of this manuscript.

## Acknowledgements

We acknowledge the MRI imaging facility, member of the France-BioImaging national infrastructure supported by the French National Research Agency (ANR-10-INBS-04, "Investments for the future"), and PHIV-La Gaillarde facility.

**Authors contribution.** A.L., T.C.X., S.T. and C.C. wrote the manuscript. T.C.X. made the Figure and Graphical Abstract.

**Financial support statement.** This work was supported by the French Agence Nationale pour la Recherche (Grant Number 2019-CE20-0009), The European Union's Horizon 2020 research and innovation programme (Grant agreement Number 857251-BIOPOLIS), the French Centre national de la Recherche Scientifique (MITI Metallomix Grant 2021 DesciFer), BioCampus Montpellier (Grant 2022 IronSEED).

**Competing interest.** None.

**Open peer review.** To view the open peer review materials for this article, please visit http://doi.org/10.1017/qpb.2025.11.

## References

Alcon, C., Comte, A., Curie, C., & Xiong, T. C. (2024). Imaging of labile Fe2+ and Fe3+ in living Arabidopsis thaliana roots. *Plant Physiology*, *kiae221*, https://doi.org/10.1093/plphys/kiae221

Bienfait, H. F., van den Briel, W., & Mesland-Mul, N. T. (1985). Free space iron pools in roots: Generation and mobilization. *Plant Physiology*, *78*(3), 596-600. https://doi.org/10.1104/pp.78.3.596

Briat, J.-F., Duc, C., Ravet, K., & Gaymard, F. (2010). Ferritins and iron storage in plants. *Ferritin: Structures, Properties and Applications*, **1800**(8), 806–814. https://doi.org/10.1016/j.bbagen.2009.12.003

Brun, A., Smokvarska, M., Wei, L., Chay, S., Curie, C., & Mari, S. (2022). MCO1 and MCO3, two putative ascorbate oxidases with ferroxidase activity, new candidates for the regulation of apoplastic iron excess in Arabidopsis. *Plant Direct*, **6**(11), e463. https://doi.org/10.1002/pld3.463

Castaings, L., Caquot, A., Loubet, S., & Curie, C. (2016). The high-affinity metal transporters NRAMP1 and IRT1 team up to take up iron under sufficient metal provision. *Scientific Reports*, **6**(1), 37222. https://doi.org/10.1038/srep37222

Clúa, J., Montpetit, J., Jimenez-Sandoval, P., Naumann, C., Santiago, J., & Poirier, Y. (2024). A CYBDOM protein impacts iron homeostasis and primary root growth under phosphate deficiency in Arabidopsis. *Nature Communications*, **15**(1), 423. https://doi.org/10.1038/s41467-023-43911-x

De Brier, N., Gomand, S. V., Donner, E., Paterson, D., Smolders, E., Delcour, J. A., & Lombi, E. (2016). Element distribution and iron speciation in mature wheat grains (Triticum aestivum L.) using synchrotron X-ray fluorescence microscopy mapping and X-ray absorption near-edge structure (XANES) imaging. *Plant, Cell & Environment*, **39**(8), 1835–1847. https://doi.org/10.1111/pce.12749

Divol, F., Couch, D., Conéjéro, G., Roschztardtz, H., Mari, S., & Curie, C. (2013). The Arabidopsis YELLOW STRIPE LIKE4 and 6 transporters control iron release from the chloroplast. *The Plant Cell*, **25**(3), 1040–1055. https://doi.org/10.1105/tpc.112.107672

Drakakaki, G., van de Ven, W., Pan, S., Miao, Y., Wang, J., Keinath, N. F., Weatherly, B., Jiang, L., Schumacher, K., Hicks, G., & Raikhel, N. (2012).
Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis. *Cell Research*, **22**(2), 413-424. https://doi.org/10.1038/cr.2011.129

Duy, D., Stübe, R., Wanner, G., & Philippar, K. (2011). The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron. *Plant Physiology*, **155**(4), 1709-1722. https://doi.org/10.1104/pp.110.170233

Duy, D., Wanner, G., Meda, A. R., von Wiren, N., Soll, J., & Philippar, K. (2007). PIC1, an ancient permease in Arabidopsis chloroplasts, mediates iron transport. *The Plant Cell*, **19**(3), 986-1006. https://doi.org/10.1105/tpc.106.047407

Eitinger, T., Suhr, J., Moore, L., & Smith, J. A. C. (2005). Secondary transporters for nickel and cobalt ions: Theme and variations. *Biometals*, **18**, 399-405. https://doi.org/10.1007/s10534-005-3714-x

Eroglu, S., Giehl, R. F., Meier, B., Takahashi, M., Terada, Y., Ignatyev, K., Andresen, E., Küpper, H., Peiter, E., & Von Wiren, N. (2017). Metal tolerance protein 8 mediates manganese homeostasis and iron reallocation during seed development and germination. *Plant Physiology*, **174**(3), 1633-1647. https://doi.org/10.1104/pp.16.016164

Giehl, R. F. H., Flis, P., Fuchs, J., Gao, Y., Salt, D. E., & von Wirén, N. (2023). Cell type-specific mapping of ion distribution in *Arabidopsis thaliana* roots. *Nature Communications*, **14**(1), 3351. https://doi.org/10.1038/s41467-023-38880-0

Hilo, A., Shahinnia, F., Druege, U., Franken, P., Melzer, M., Rutten, T., von Wirén, N., & Hajirezaei, M.-R. (2017). A specific role of iron in promoting meristematic cell division during adventitious root formation. *Journal of Experimental Botany*, **68**(15), 4233-4247. https://doi.org/10.1093/jxb/exr248

Hoang, M. T. T., Almeida, D., Chay, S., Alcon, C., Corratge-Faillie, C., Curie, C., & Mari, S. (2021). AtDTX25, a member of the multidrug and toxic compound extrusion family, is a vacuolar ascorbate transporter that controls intracellular iron cycling in Arabidopsis. *New Phytologist*, **231**(5), 1956-1967. https://doi.org/10.1111/nph.17526

Ideas, M. A., Grant-Grant, S., Navarro, N., Perez, M. F., & Roschztardtz, H. (2017). Dynamic subcellular localization of iron during embryo development in Brassicaceae seeds. *Frontiers in Plant Science*, **8**, 2186. https://doi.org/10.3389/fpls.2017.02186

Jeong, J., Cohu, C., Kerkeb, L., Pilon, M., Connolly, E. L., & Guerinot, M. L. (2008). Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions. *Proceedings of the National Academy of Sciences*, **105**(30), 10619-10624. https://doi.org/10.1073/pnas.0708367105

Jiménez-Lamana, J., Bierla, K., Leskova, A., Curie, C., Szpunar, J., & Mari, S. (2023). Single cell ICP-MS as a powerful analytical tool to determine metal content in individual pollen grains. *Journal of Analytical Atomic Spectrometry*, **38**(8), 1560-1563. https://doi.org/10.1039/D3JA00140G

Kim, L. J., Tsuyuki, K. M., Hu, F., Park, E. Y., Zhang, J., Iraheta, J. G., Chia, J., Huang, R., Tucker, A. E., & Clyne, M. (2021). Ferroportin 3 is a dual-targeted mitochondrial/chloroplast iron exporter necessary for iron homeostasis in Arabidopsis. *The Plant Journal*, **107**(1), 215-236. https://doi.org/10.1111/tpj.15286

Kim, S. A., Punshon, T., Lanzirotti, A., Li, L., Alonso, J. M., Ecker, J. R., Kaplan, J., & Guerinot, M. L. (2006). Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1. *Science*, **314**(5803), 1295-1298. https://doi.org/10.1126/science.1132563

Koppenol, W. H., & Hider, R. H. (2019). Iron and redox cycling. Does and don'ts. *Iron as Soul of Life on Earth Revisited: From Chemical Reaction, Ferroptosis to Therapeutics*, **133**, 3-10. https://doi.org/10.1016/j.freeradbiomed.2018.09.022

Kroh, G. E., & Pilon, M. (2020). Regulation of iron homeostasis and use in chloroplasts. *International Journal of Molecular Sciences*, **21**(9), 3395. https://doi.org/10.3390/ijms21093395

Lanquar, V., Lelièvre, F., Bolte, S., Hamès, C., Alcon, C., Neumann, D., Vansuyt, G., Curie, C., Schröder, A., & Krämer, U. (2005). Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. *The EMBO Journal*, **24**(23), 4041-4051. https://doi.org/10.1038/sj.emboj.7600864

Liu, X. X., Zhu, X. F., Zheng, S. J., & Jin, C. W. (2023). Beyond iron-storage pool: Functions of plant apoplastic iron during stress. *Trends in Plant Science*, **28**(8), 941-954. https://doi.org/10.1016/j.tplants.2023.03.007

López-Millán, A. F., Duy, D., & Philippar, K. (2016). Chloroplast iron transport proteins – Function and impact on plant physiology. *Frontiers in Plant Science*, **7**, 178. https://doi.org/10.3389/fpls.2016.00178

Lott, J. N., Ockenden, I., Raboy, V., & Batten, G. D. (2001). A global estimate of phytic acid and phosphorus in crop grains, seeds, and fruits. In *Food phytates* (pp. 23–40). CRC Press

Maniero, R. A., Picco, C., Hartmann, A., Engelberger, F., Gradogna, A., Scholz-Starke, J., Melzer, M., Künze, G., Carpaneto, A., Von Wirén, N., & Giehl, R. F. H. (2024). Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency. *Nature Communications*, **15**(1), 422. https://doi.org/10.1038/s41467-023-43912-w

Martín-Barranco, A., Spielmann, J., Dubeaux, G., Vert, G., & Zelazny, E. (2020). Dynamic control of the high-affinity iron uptake complex in root epidermal cells. *Plant Physiology*, **184**(3), 1236-1250. https://doi.org/10.1104/pp.20.00234

Miyashita, S., Groombridge, A. S., Fujii, S., Minoda, A., Takatsu, A., Hioki, A., Chiba, K., & Inagaki, K. (2014). Highly efficient single-cell analysis of microbial cells by time-resolved inductively coupled plasma mass spectrometry. *Journal of Analytical Atomic Spectrometry*, **29**(9), 1598-1606. https://doi.org/10.1039/C4JA00040D

Montacié, C., Riondet, C., Wei, L., Darrière, T., Weiss, A., Pontvianne, F., Escande, M.-L., de Bures, A., Jobet, E., Barbarossa, A., Carpentier, M.-C., Aarts, M. G. M., Attina, A., Hirtz, C., David, A., Marchand, V., Motorin, Y., Curie, C., Mari, S., & Sáez-Vásquez, J. (2023). NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis. *Journal of Experimental Botany*, **74**(15), 4384-4400. https://doi.org/10.1093/jxb/erad180

Müller, J., Toev, T., Heisters, M., Teller, J., Moore, K. L., Hause, G., Dinesh, D. C., Bürstenbinder, K., & Abel, S. (2015). Iron-dependent callose deposition adjusts root meristem maintenance to phosphate availability. *Developmental Cell*, **33**(2), 216-230. https://doi.org/10.1016/j.devcel.2015.02.007

Naumann, C., Heisters, M., Brandt, W., Janitza, P., Alfs, C., Tang, N., Toto Nienguesso, A., Ziegler, J., Imre, R., Mechtler, K., Dagdas, Y., Hoehenwarter, W., Sawers, G., Quint, M., & Abel, S. (2022). Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development. *Current Biology*, **32**(10), 2189.e6-2205.e6. https://doi.org/10.1016/j.cub.2022.04.005

Peng, J.-S., Zhang, B.-C., Chen, H., Wang, M.-Q., Wang, Y.-T., Li, H.-M., Cao, S.-X., Yi, H.-Y., Wang, H., & Zhou, Y.-H. (2021). Galactosylation of rhamnogalacturonan-II for cell wall pectin biosynthesis is critical for root apoplastic iron reallocation in Arabidopsis. *Molecular Plant*, **14**(10), 1640-1651. https://doi.org/10.1016/j.molp.2021.06.016

Persson, D. P., Chen, A., Aarts, M. G. M., Salt, D. E., Schjoerring, J. K., & Husted, S. (2016). Multi-element bioimaging of Arabidopsis thaliana roots. *Plant Physiology*, **172**(2), 835-847. https://doi.org/10.1104/pp.16.00770

Platre, M. P., Satbhai, S. B., Brent, L., Gleason, M. F., Cao, M., Grison, M., Glavier, M., Zhang, L., Gaillochet, C., Goeschl, C., Giovannetti, M., Enugutti, B., Neveu, J., von Reth, M., Alcázar, R., Parker, J. E., Vert, G., Bayer, E., & Busch, W. (2022). The receptor kinase SRF3 coordinates iron-level and flagellin dependent defense and growth responses in plants. *Nature Communications*, **13**(1), 4445. https://doi.org/10.1038/s41467-022-32167-6

Punshon, T., Hirschi, K., Yang, J., Lanzirotti, A., Lai, B., & Guerinot, M. L. (2012). The role of CAX1 and CAX3 in elemental distribution and abundance in Arabidopsis seed. *Plant Physiology*, **158**(1), 352-362. https://doi.org/10.1104/pp.111.184812

Ravet, K., Touraine, B., Boucherez, J., Briat, J.-F., Gaymard, F., & Cellier, F. (2009). Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis. *The Plant Journal*, **57**(3), 400-412. https://doi.org/10.1111/j.1365-313X.2008.03698.x

Roschztardtz, H., Conéjéro, G., Curie, C., & Mari, S. (2009). Identification of the endodermal vacuole as the iron storage compartment in the Arabidopsis
embryo. *Plant Physiology*, **151**(3), 1329-1338. https://doi.org/10.1104/pp.
109.144444

Roschztardtz, H., Conéjéro, G., Divol, F., Alcon, C., Verdeil, J.-L., Curie, C.,
& Mari, S. (2013). New insights into Fe localization in plant tissues. *Frontiers
in Plant Science*, **4**, 350. https://doi.org/10.3389/fpls.2013.00350

Roschztardtz, H., Grillet, L., Isaure, M.-P., Conéjéro, G., Ortega, R., Curie,
C., & Mari, S. (2011). Plant cell nucleolus as a hot spot for iron. *Journal
of Biological Chemistry*, **286**(32), 27863-27866. https://doi.org/10.1074/jbc.
C111.269720

Roschztardtz, H., Séguéla-Arnaud, M., Briat, J.-F., Vert, G., & Curie, C. (2011). The FRD3 citrate effluxer promotes iron nutrition between symptomatically disconnected tissues throughout Arabidopsis development. *The Plant Cell*, **23**(7), 2725-2737. https://doi.org/10.1105/tpc.111.088088

Shen, X., Zhang, H., He, X., Shi, H., Stephan, C., Jiang, H., Wan, C., & Eichholz, T. (2019). Evaluating the treatment effectiveness of copper-based algaecides on toxic algae Microcystis aeruginosa using single cell-inductively coupled plasma-mass spectrometry. *Analytical and Bioanalytical Chemistry*, **411**(21), 5531-5543. https://doi.org/10.1007/s00216-019-01933-9

Sheraz, S., Wan, Y., Venter, E., Verma, S. K., Xiong, Q., Waites, J., Connorton, J. M., Shewry, P. R., Moore, K. L., & Balk, J. (2021). Subcellular dynamics studies of iron reveal how tissue-specific distribution patterns are established in developing wheat grains. *New Phytologist*, **231**(4), 1644-1657. https://doi.org/10.1111/nph.17440

Shikanai, T., Müller-Moulé, P., Munekage, Y., Niyogi, K. K., & Pilon, M. (2003). PAA1, a P-type ATPase of Arabidopsis, functions in copper transport in chloroplasts. *The Plant Cell*, **15**(6), 1333-1346. https://doi.org/10.1105/tpc.011817

Singh, S. P., Vogel-Mikuš, K., Vavpetič, P., Jeromel, L., Pelicon, P., Kumar, J., & Tuli, R. (2014). Spatial X-ray fluorescence micro-imaging of minerals in grain tissues of wheat and related genotypes. *Planta*, **240**(2), 277-289. https://doi.org/10.1007/s00425-014-2084-4

Strasser, O., Köhl, K., & Römheld, V. (1999). Overestimation of apoplastic Fe in roots of soil grown plants. *Plant and Soil*, **210**, 179-189. https://doi.org/10.1023/A:1004650506592

Teng, Y.-S., Su, Y., Chen, L.-J., Lee, Y. J., Hwang, I., & Li, H. (2006). Tic21 is an essential translocon component for protein translocation across the chloroplast inner envelope membrane. *The Plant Cell*, **18**(9), 2247-2257. https://doi.org/10.1105/tpc.106.044305

Vélez-Bermúdez, I. C., & Schmidt, W. (2023). Iron sensing in plants. *Frontiers in Plant Science*, **14**, 1145510. https://doi.org/10.3389/fpls.2023.1145510

Vigani, G., Bohic, S., Faoro, F., Vekemans, B., Vincze, L., & Terzano, R. (2018). Cellular fractionation and nanoscopic X-ray fluorescence imaging analyses reveal changes of zinc distribution in leaf cells of iron-deficient plants. *Frontiers in Plant Science*, **9**, 1112. https://doi.org/10.3389/fpls.2018.01112

Xing, Y., Xu, N., Bhandari, D. D., Lapin, D., Sun, X., Luo, X., Wang, Y., Cao, J., Wang, H., Coaker, G., Parker, J. E., & Liu, J. (2021). Bacterial effector targeting of a plant iron sensor facilitates iron acquisition and pathogen colonization. *The Plant Cell*, **33**(6), 2015-2031. https://doi.org/10.1093/plcell/koab075

Xu, Z.-R., Cai, M.-L., Yang, Y., You, T.-T., Ma, J. F., Wang, P., & Zhao, F.-J. (2022). The ferroxidases LPR1 and LPR2 control iron translocation in the xylem of Arabidopsis plants. *Molecular Plant*, **15**(12), 1962-1975. https://doi.org/10.1016/j.molp.2022.11.003