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. 2026 Feb 26;49(6):3290–3305. doi: 10.1111/pce.70463

Arbuscular Mycorrhiza Modulates Iron Distribution and Vacuolar Iron Transporter Expression in Tomato, Whereas Iron Limitation Reduces Mycorrhization

Víctor M López‐Lorca 1, Olga López‐Castillo 1, Mª Jesús Molina‐Luzón 1, Nuria Ferrol 1,✉
PMCID: PMC13136555  PMID: 41744165

ABSTRACT

Plants have evolved highly efficient strategies to maintain iron (Fe) homeostasis. In this study, we investigate the impact of arbuscular mycorrhizal (AM) symbiosis on the Fe‐deficiency response and ionomic profile of tomato plants, as well as how Fe availability affects AM symbiosis. Fe deficiency and AM colonization both reduced shoot Fe concentrations, while root Fe concentrations increased in AM plants. Notably, Fe accumulated in cortical cells colonized by arbuscules. We further show that Fe deficiency reduces expression of AM‐related tomato genes (SlEXO84, SlRAM1, SlAMT2.2 and SlPT4) and of the fungal RiEF1α gene. These findings indicate that Fe availability is crucial for sustaining AM colonization and symbiotic functionality. Under Fe‐limiting conditions, AM symbiosis enhances the Strategy I Fe acquisition pathway (SlFRO1, SlIRT1), an effect not observed under Fe‐sufficient conditions. Four vacuolar transporter genes of the VIT/VTL family were identified in the tomato genome. Yeast complementation assays revealed that SlVIT1, SlVTL1, and SlVTL2 function as dual Fe/Mn transporters, whereas SlVIT2 appears to function as a Mn transporter. The high Fe demand of AM symbiosis is supported by the reduced expression of SlVIT1 and SlVTL1 in mycorrhizal roots. Ionomic analysis shows that AM colonization partially alleviates Fe deficiency‐induced nutrient imbalances, highlighting its contribution to improved mineral homeostasis under Fe stress.

Summary statement

  • We report for the first time that iron accumulates in arbuscule‐containing cells in mycorrhizal tomato roots and that iron is crucial for arbuscular mycorrhiza.

  • The symbiosis modulates plant responses to iron deficiency and buffers ionomic imbalances.

1. Introduction

Iron (Fe) is an essential micronutrient required for the proper development and survival of all organisms. It plays structural roles in proteins and functions as a cofactor for various enzymes. In plants, Fe is crucial for key physiological processes, such as photosynthesis, mitochondrial respiration and nitrogen metabolism (Connorton et al. 2017). Although Fe is abundant in nature, it is often poorly available to plants, particularly in alkaline soils, because it predominantly exists in its oxidized state Fe (III). Fe deficiency leads to chlorosis and poor plant growth and development (Kabir et al. 2013). Consequently, plants have evolved highly efficient strategies to acquire Fe from the rhizosphere (Grotz and Guerinot 2006). All plant species except grasses utilize Strategy I for Fe uptake, involving rhizosphere acidification by a plasma membrane H+‐ATPase, Fe (III) reduction by a ferric reductase (FRO1) and Fe (II) uptake via specific transporters (IRT and NRAMP1). Grasses employ a Strategy II, releasing phytosiderophores to the rhizosphere that chelate Fe (III), with uptake mediated by oligopeptide transporters of the YS1 or YS1‐like family (Liang 2022). After absorption, Fe is transported within the symplast primarily as Fe (II)‐nicotianamine complexes. At the cellular level, Fe is either incorporated into metalloproteins or compartmentalized into the vacuoles through transporters of the vacuolar iron transporter (VIT) family (Ram et al. 2021).

Besides these intrinsic Fe uptake mechanisms, plants can cope with Fe deficiency by associating with beneficial soil microorganisms (Marschner et al. 2011; Lurthy et al. 2021). Siderophore‐mediated Fe acquisition by rhizosphere microorganisms is well documented (Lurthy et al. 2020); however, the role of arbuscular mycorrhizal (AM) fungi, one of the most prominent groups of soil symbionts involved in plant mineral nutrition, remains largely unexplored in the context of Fe acquisition.

AM fungi, belonging to the subphylum Glomeromycotina, form mutualistic associations with most terrestrial plant species (Azcón‐Aguilar and Barea 2015). They colonize the root cortical cells and develop an extensive network of extraradical hyphae in the surrounding soil, enhancing nutrient uptake beyond root depletion zones. Nutrients are delivered to the plant via arbuscules, highly branched structures formed within cortical cells, while the fungus receives photosynthates in return (Smith and Read 2008; Duan et al. 2024). While the primary benefit of AM symbiosis is an improved phosphorus acquisition, root colonization by AM fungi has also been shown to enhance the uptake of micronutrients, particularly under nutrient‐deficient conditions (Ferrol et al. 2016; Moreno Jiménez et al. 2024). The role of AM fungi in plant Zn and Cu nutrition is well established. In contrast, their impact on Fe nutrition is more variable and often inconsistent (Clark and Zeto 2000), depending on several factors, such as plant growth conditions, host species, and the specific AM fungal taxa involved (Caris et al. 1998; Kobae et al. 2014; Lehmann and Rillig 2015; Suzuki et al. 2000). Recent evidence has provided insights into the molecular mechanisms by which AM fungi alleviate Fe deficiency in host plants (Liu et al. 2023; Rajapitamahuni et al. 2023). Increased Fe uptake under Fe‐limiting conditions in mycorrhizal plants may result from enhanced Fe bioavailability in the rhizosphere and the up‐regulation of genes involved in Fe acquisition. Evidence includes increased phytosiderophore release in Strategy II plants (Prity et al. 2020), elevated ferric chelate reductase activity in various Strategy I plants (Kabir et al. 2020), and induction of genes such as IRT1, FRO1, NAS, YS1, and OPT8 in various species (Kobae et al. 2014; Kabir et al. 2020; Prity et al. 2020; Rahman et al. 2020; Rahimi et al. 2021).

Despite recent progress, the molecular mechanisms underlying AM‐mediated Fe acquisition are still largely unclear. To gain further insights into Fe homeostasis in the context of AM, this study analyzed the impact of AM on Fe nutrition and the Fe‐deficiency response in tomato plants. Tomato, a Strategy I plant with high agronomic and nutritional value and particular sensitivity to Fe deficiency—especially in calcareous soils—was selected for this study not only for its economic importance, but also because it serves as a model species for studying Fe homeostasis in Strategy I plants (Zamboni et al. 2012) and AM symbiosis (Ho‐Plágaro et al. 2018). Additionally, considering the reported cross‐talk between Fe and other mineral nutrients (Fan et al. 2021; Rai et al. 2021), this study also aimed to analyze how AM symbiosis influences the tomato ionome under Fe deficiency.

2. Materials and Methods

2.1. Biological Materials and Growth Conditions

Tomato seeds (Solanum lycopersicum cv. Moneymaker) were surface sterilized with 5% commercial bleach for 3 min, washed with sterile water and maintained in sterile water for 2 h. Afterwards seeds were germinated under sterile conditions on wet filter paper in Petri plates in darkness at 25°C for 4 d. Germinated seeds were transferred to pots containing 1.6 L of sterilized sand previously washed with 0.03 M HCl to eliminate metal traces and then rinsed with distilled water until pH 7. The AM fungal inoculum used was Rhizophagus irregularis Schenck and Smith DAOM197189 grown in monoxenic cultures. The inoculum was prepared by blending the medium with sterile 2.5 mM citrate buffer (pH 6.0). Seedlings were inoculated by adding to the growth substrate 3 mL of the AM fungal inoculum containing 1650 spores (mycorrhizal treatment). Non‐mycorrhizal plants were prepared by adding 3 mL of an inoculum filtrate. Plants were developed in a growth chamber under 16 h light (24°C)/8 h dark (20°C). They were watered twice a week with a modified half‐strength Hoagland solution containing 0.125 mM KH2PO4 and 50 µM EDTA‐Fe(III) (control treatment) or without EDTA‐Fe(III) (Fe deficiency treatment). Each treatment consisted of seven replicates. Plants were harvested 12 weeks after inoculation. At harvesting, plant biomass was determined by measuring root and shoot fresh weights. An aliquot of each root system was separated to estimate mycorrhizal colonization. Roots and shoots were frozen in liquid nitrogen and stored at −80°C until used.

The ccc1Δ and pmr1Δ mutants of Saccharomyces cerevisiae were used in this work. ccc1Δ lacks the vacuolar membrane transporter Ccc1 (Li et al. 2001) and pmr1Δ is unable to transport Mn into Golgi vesicles and cannot withstand toxic levels of the metal (Lapinskas et al. 1995). Yeast cells were grown on YPD or minimal synthetic dextrose (SD) medium, supplemented with the appropriate amino acids.

2.2. Elemental Analyses

An aliquot of the shoot and root samples of each plant was oven‐dried at 65°C for 2 days, ground to a fine powder, ashed at 550°C and digested in H2O/HNO3. Tissue nutrient concentrations were analyzed by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP‐OES; ICP 6500 Duo Thermo) analysis in the Instrumentation Facility of the Estación Experimental del Zaidín, EEZ‐CSIC, Granada, Spain. The Fe translocation factor was calculated as the ratio of Fe concentration in the shoot to that in the root.

2.3. Perls Iron Staining of Root Transversal Sections

Roots of non‐mycorrhizal and mycorrhizal plants were washed with Milli‐Q water and blotted dry with filter paper. Then, root segments (1 cm) from the middle of the root system were vacuum infiltrated with 4% (w/v) paraformaldehyde and 0.2% (v/v) glutaraldehyde in a 100 mM sodium cacodylate buffer (pH 7.2) for 2 h and incubated for 16 h in the same solution. The fixed samples were washed with 0.05 M Na‐cacodylate buffer (pH 7.2) three times, and dehydrated in successive baths of 30%, 50%, 70%, 90%, and 100% ethanol. Next, the roots were embedded in the LR‐GOLD resin, according to the manufacturer's instructions, and thin transversal Section (1 μm) were obtained using a microtome. The sections were deposited on glass slides and incubated at room temperature for 45 min in Perls stain solution containing 4% (v/v) HCl and 4% (w/v) K‐ferrocyanide, following the procedure described by Ivanov et al. (2014). Negative controls were prepared by incubating the sections with 4% (v/v) HCl only. Finally, the sections were washed three times with Milli‐Q water and bright images were acquired at the Microscopy Service (CETEM) of the Estación Experimental del Zaidin using a Leica DMI600B microscope equipped with a high‐resolution digital camera.

2.4. RNA Extraction and cDNA Synthesis

Total RNA was extracted from tomato roots using the phenol/SDS method followed by LiCl precipitation (García‐Rodríguez et al. 2007). RNAs were treated with DNase using the RNA‐free DNase set (PROMEGA) using the manufacturer's protocol. cDNAs were synthetized from 1 µg of total DNase‐treated RNA in a 20 µL reaction using Super‐Script IV Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions.

2.5. Gene Expression Analyses

Gene expression was analyzed by real‐time RT‐PCR using a QuantStudio 3 (Applied Biosystem) in the synthetized cDNAs. Each 12 µL reaction contained 1 µl of a 1:10 dilution of cDNA, 0.5 µL 10 mM each primer and 6 µL iTaq (Bio‐Rad). The primer pairs used for qRT‐PCR are presented in Supporting Information S1: Table 1. The specificity of the primer sets was analyzed by PCR amplification of the R. irregularis cDNA. The real‐time PCR program consisted of an initial incubation at 95°C for 30 s, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s, where the fluorescence signal was measured, and a final step with a heat‐dissociation protocol to check the specificity of PCR amplification procedure. Efficiency of the different primer pairs was determined through a real‐time PCR on several dilutions of cDNA. The results obtained for the different treatments were standardized to the expression levels of SlEF1α and SlACTIN. Real‐time RT‐PCR determinations were carried out on at least three independent biological samples. Real time RT‐PCR reactions were performed at least two times for each biological sample, with the threshold cycle (Ct) determined in duplicate. Relative expression levels were calculated using the 2−∆Ct method (Schmittgen and Livak 2008) and the standard error was computed from the average of the ΔCt values for each biological sample.

2.6. SlVIT Sequence Identification and Analyses

SlVIT paralog sequences (Supporting Information S1: Table 2) were selected through a Blastp search in NCBI (National Center for Biotechnology Information; https://www.ncbi.nlm.nih.gov/) database using the SlVIT sequence deposited in the plant membrane protein database Aramemnon (http://aramemnon.uni-koeln.de/) as template. Arabidopsis thaliana VIT/VTL protein sequences were retrieved from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/). Conserved Domain Database at NCBI was used to identify conserved domains of proteins. Primer3 (https://primer3.ut.ee/) was used to design the gene‐specific primers. Potential transmembrane domains were predicted using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM). Structural models of the proteins were generated using MyDomains tool of Prosite (https://prosite.expasy.org/mydomains/). 3D models were predicted using the Phyre2 software (http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index) based on the crystal structure of iron transporter VIT1 with cobalt ion of Eucalyptus grandis (ID: c6iu3A). The alignment of the putative amino acid sequences of the SlVIT/VTL were carried out using Mega‐X. Homology and similarity percentages were performed with SIAS (http://imed.med.ucm.es/Tools/sias.html). Mega‐X was used to calculate phylogenetic relationships with the Neighbour‐Joining method.

2.7. Heterologous Expression

The open reading frames of the S. lycopersicum VIT genes were amplified from cDNA extracted from tomato roots by PCR using the corresponding primers pairs (Supporting Information S1: Table 1) and cloned into the expression vector pDRF1‐GW using the Gateway technology (Invitrogen). To be recognized by de BP enzyme, full‐length cDNA sequences were flanked with the sequences attB1 and attB2. PCR products were cloned following manufacturer's instructions. All constructs were verified by sequencing. S. cerevisiae mutant strains ccc1Δ and pmr1Δ were transformed with the different pDRf1‐VIT constructs or with the empty vector using a lithium acetate‐based method (Schiestl and Gietz 1989). Yeast transformants were selected in SD medium by uracil autotrophy. For drop tests, yeast transformants were harvested by centrifugation from a liquid culture grown to exponential phase in SD medium without uracil, washed three times with milli‐Q H2O and adjusted to a final OD600 of 1. Then, 5 µL of serial 1:10 dilutions were spotted on the corresponding selective medium. ccc1Δ transformants were spotted onto SD‐URA supplemented or not with 5 mM FeSO4. pmr1Δ transformed mutants were spotted onto SD without uracil supplemented or not with 2 mM MnCl2.

2.8. Statistical Analyses

Statgraphics Centurion XVI software was used for the statistical analysis of means and standard error determinations. Data were subjected to a two‐way ANOVA, considering mycorrhization and Fe as factors. Post hoc comparisons were evaluated using the Duncan's test to find differences among groups of means (p < 0.05), when necessary. All analyses were based on at least three biological replicates for treatment. Nutrients data were logarithm transformed and subjected to a sparse partial least‐squares discriminant analysis (sPLSDA) by using METABOANALIST (https://www.metaboanalyst.ca/) web‐based metabolomic package.

3. Results

3.1. Fe Deficiency and Mycorrhizal Colonization Reduce Root‐to‐Shoot Fe Translocation

Shoot fresh weight was not affected by either Fe deficiency or mycorrhizal colonization. However, root fresh weight increased significantly under Fe deficiency in non‐mycorrhizal plants (Figure 1a). Shoot Fe concentration decreased in plants supplied with an Fe‐free nutrient solution (Figure 1b). No significant differences were found in root Fe concentration between Fe‐sufficient and ‐deficient conditions. Compared to non‐inoculated plants, shoot Fe concentration was lower in mycorrhizal plants under Fe‐sufficient conditions (Figure 1b). In contrast, root Fe concentration markedly increased in mycorrhizal plants under both Fe treatments. Consequently, the Fe translocation factor decreased due to both Fe deficiency and mycorrhizal colonization (Figure 1c).

Figure 1.

Figure 1

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Impact of Fe availability and mycorrhizal colonization on tomato biomass, Fe concentration and translocation factor. (a) Shoot and root fresh weight of mycorrhizal (Myc) and non‐mycorrhizal (NM) plants fertilized with a half‐strength Hoagland solution containing 50 µM Fe (Control) or with a modified nutrient solution lacking Fe (‐Fe). (b) Shoot and root Fe concentration of mycorrhizal (Myc) and non‐mycorrhizal (NM) plants under Fe‐sufficient (Control) and Fe‐deficient (‐Fe) conditions. (c) Fe translocation factor in mycorrhizal (Myc) and non‐mycorrhizal (NM) plants under Fe‐sufficient (Control) and Fe‐deficient (‐Fe) conditions. Values represent means ± standard error (n = 7). Different letters indicate statistically significant differences among treatments (p < 0.05) according to Duncan's test.

3.2. Cortical Arbuscule‐Colonized Cells as Sites of Iron Accumulation in Mycorrhizal Roots

The high levels of Fe detected in the roots of mycorrhizal plants prompted us to investigate its distribution through histochemical analysis using Perls staining on root cross‐sections (Figure 2). A blue Perls precipitate was observed in the cortical cells of mycorrhizal roots containing arbuscules (Figure 2d), indicating that the reduced Fe translocation in these plants was due, at least partially, to its accumulation in the fungal structures. No signal was detected in mycorrhizal root sections incubated with HCl (negative control, Figure 2c) or in non‐mycorrhizal roots treated with the Perls staining solution (Figure 2b).

Figure 2.

Figure 2

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Histochemical Perls staining of transversal root sections of non‐mycorrhizal (NM, a and b) and mycorrhizal (Myc, c and d) plants, incubated either with 4% (v/v) HCl (unstained control, a and c) or the Perls reagent containing 4% (v/v) HCl and 4% (w/v) K‐ferrocyanide (stained, b and d) for 45 min. Cortical cells containing arbuscules in mycorrhizal roots (d) show a blue Perls precipitate indicative of Fe accumulation. The scale bars correspond to 100 μm. The images shown are representative of ten sections analysed from three independent plants per treatment.

3.3. Fe Deficiency Down‐Regulates Mycorrhiza‐Related Genes

To investigate the impact of Fe deficiency on AM, the expression of a panel of AM‐marker genes was analyzed, including SlRAM1 (SolDB accession Solyc02g094340.1), SlEXO84 (SolDB accession Solyc09g072720.2), SlAMT2.2 (SolDB accession Solyc08g067080.1) and SlPT4 (SolDB accession Solyc06g051850.1) (Ho‐Plágaro et al. 2021). SlRAM1 is a GRAS transcription factor that regulates the expression of genes involved in arbuscule development, such as SlEXO1 playing a role in periarbuscular membrane formation, and nutrient transporters like SlPT4 and SlAMT2.2. which mediate phosphate and ammonium uptake by the periarbucular membrane, respectively. The expression of all analyzed AM‐related genes was significantly down‐regulated under Fe deficient conditions (Figure 3). The abundance of the fungus in the roots was also estimated by measuring the expression levels of the constitutively expressed fungal gene RiEF1α. A threefold reduction in RiEF1α expression was detected in mycorrhizal roots of plants grown under Fe‐deficient conditions.

Figure 3.

Figure 3

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Effect of Fe deficiency on the expression of mycorrhizal marker genes in tomato mycorrhizal roots. Plants were fertilized with a half‐strength Hoagland solution containing 50 µM Fe (Control) or with a modified solution lacking Fe (‐Fe). Gene expression was analysed in RNA extracts and calculated using the 2‐∆CT method with SlEF1α and SlACTIN as normalizers. Bars represent means ± standard error (n = 3). Asterisks indicate statistically significant differences between treatments (p < 0.05; Student's t‐test).

3.4. Expression Patterns of Fe‐Deficiency Response Genes

To assess the impact of AM on the Fe‐deficiency response in tomato, the expression patterns of previously reported tomato genes involved in Fe uptake and distribution were analyzed by qRT‐PCR in roots of non‐mycorrhizal and mycorrhizal plants grown under Fe‐sufficient and Fe‐deficient conditions. Transcripts of SlFRO1, the first component of the Strategy I Fe acquisition system, were not detected in roots of plants grown under Fe‐sufficient conditions, likely due to expression levels being below the detection threshold. Under Fe‐deficient conditions, SlFRO1 expression was significantly up‐regulated, with a stronger induction observed in mycorrhizal roots. Expression of SlIRT1, the Fe permease and second component of the Strategy I, was not significantly affected by Fe‐deficiency in non‐mycorrhizal roots. However, SlIRT1 was up‐regulated in mycorrhizal roots under Fe‐deficient conditions. Under Fe‐sufficient conditions, SlIRT1 transcript levels were lower in mycorrhizal roots compared to non‐mycorrhizal roots (Figure 4).

Figure 4.

Figure 4

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Effect of Fe‐deficiency and mycorrhizal colonization on the expression of Fe deficiency‐responsive genes in tomato roots. Gene expression was analysed in RNAs isolated from Solanum lycopersicum non‐mycorrhizal (NM) and mycorrhizal (Myc) roots of plants fertilized with half‐strength Hoagland solution containing 50 µM Fe (Control) or a modified nutrient solution lacking Fe (‐Fe). Relative expression was calculated, using the 2‐∆CT method with SlEF1α and SlACTIN as normalizers. Bars represent means ± standard error (n = 3). Different letters indicate statistically significant differences among treatments (p < 0.05, Duncan's test).

The expression of SlNRAMP1 and SlNRAMP3, which encode two transporters of the resistance‐associated macrophage protein (NRAMP) family with demonstrated Fe transport activity in yeast (Bereczky et al. 2003), was also assessed. SlNRAMP1 expression increased in mycorrhizal roots under Fe‐sufficient conditions. In contrast, expression of the Fe transporter SlNRAMP3 was down‐regulated by Fe‐deficiency in non‐mycorrhizal roots and by AM colonization under Fe‐sufficient conditions. In mycorrhizal roots, SlNRAMP3 expression was not affected by Fe availability (Figure 4). Finally, expression of SlCHLN, which encodes a protein involved in the synthesis of the non‐protein amino acid nicotianamine, essential key molecule for Fe distribution (Higuchi et al. 1996), was down‐regulated by Fe‐deficiency and AM colonization (Figure 4).

3.5. Fe Deficiency and AM Down‐Regulate Vacuolar Iron Transporters of the SlVIT/VTL Family

The high accumulation of Fe observed in mycorrhizal roots led us to investigate the expression patterns of genes involved in vacuolar Fe compartmentalization. Since vacuolar iron transporters of the VIT/VTL family have not yet been reported in tomato, we searched the S. lycopersicum genome for Fe transporters of this family and identified four candidate genes (SlVIT1, SlVIT2, SlVTL1 and SlVITL2), based on the predicted secondary structure of their deduced amino acid sequences (Supporting Information S2: Figure 1). Vacuolar iron Transporters Like (VTLs) are functional homologs of VITs that lack the cytosolic loop thought to mediate Fe(II)/H+ antiport in VITs (Sorribes‐Dauden et al. 2020). All four SlVIT/VTL proteins contain the conserved domains characteristic of the Ccc1/VIT1 family.

The full‐length cDNAs of SlVIT1, SlVIT2, SlVTL1 and SlVTL2 encode open reading frames of 720, 846, 648 and 654 nucleotides, respectively, corresponding to proteins of 281‐215 amino acids. All four SlVIT/VTL proteins are predicted to have five transmembrane domains, with the N‐terminus oriented toward the cytosol. Notably, SlVIT1 and SlVIT2 possess three cytosolic α‐helices between transmembrane domains 2 and 3, which, together with the C‐terminal end of the second transmembrane domain, form the metal binding domain (MBD) (Kato et al. 2019) (Supporting Information S2: Figure 1a,b). In contrast, SlVTL1 and SlVTL2 lack these cytosolic helices. Sequence similarity among the deduced amino acids ranged from 24.94% to 61.61% (Supporting Information S2: Figure 1c). A phylogenetic analysis of the VIT/VTL proteins of S. lycopersicum and Arabidopsis thaliana grouped the VIT and VTL proteins of both species into two distinct clades (Supporting Information S2: Figure 1d).

As expected for genes encoding metal transporters involved in vacuolar compartmentalization, SlVIT1, SlVIT2, SlVTL1 and SlVTL2 transcript levels were lower under Fe‐deficient conditions in roots of non‐mycorrhizal plants. SlVIT2 expression was up‐regulated in mycorrhizal roots regardless of Fe availability. Under Fe‐sufficient conditions, transcript accumulation of SlVIT1 and SlVTL1 significantly decreased in mycorrhizal roots, while SlVTL2 expression remained unaffected. In contrast, under Fe‐deficient conditions, expression of SlVIT1 and SlVTL2 expression increased in colonized roots (Figure 5).

Figure 5.

Figure 5

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Effect of Fe deficiency and mycorrhizal colonization on SlVIT/VTLs gene expression. Non‐mycorrhizal (NM) and mycorrhizal (Myc) plants were grown with either a half‐strength Hoagland solution containing 50 µM Fe (Control) or a modified nutrient solution lacking Fe (‐Fe). Relative expression levels were determined using the 2‐∆CT method, with SlEF1α and SlACTIN as normalizers. Data represent means ± standard error (n = 3). Different letters indicate statistically significant differences between treatments (p < 0.05, Duncan's test).

Next, the metal transport activity of the identified SlVIT/VTL proteins was assessed using yeast complementary assays, by testing their ability to rescue the phenotype of a yeast mutant lacking the vacuolar Fe transporter gene ccc1 (ccc1Δ). In yeast, ccc1 encodes a vacuolar transporter responsible for Fe sequestration. ccc1Δ cells are hypersensitive to high Fe concentrations due to their inability to store Fe in the vacuole (Li et al. 2001). Expression of SlVIT1, SlVTL1 and SVTL2 under the control of the PMA1 promoter restored growth of ccc1Δ cells on SD medium supplemented with 5 mM FeSO4. In contrast, cells expressing SlVIT2 or the empty vector (pDRf1‐GW) failed to grow under these conditions. These data indicate that SlVIT1, SlVTL1 and SlVTL2 cDNAs encode functional Fe transporters (Figure 6a). However, the lack of ccc1Δ complementation by SlVIT2 may be due to an artefact of the heterologous system, such as its targeting to a different subcellular membrane, and therefore a role as an Fe transporter cannot be excluded.

Figure 6.

Figure 6

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Functional analysis of Solanum lycopersicum VIT/VTLs proteins in yeast. (a) Complementation assay of the ccc1Δ yeast mutant, which is hypersensitive to high Fe concentrations due to impaired vacuolar Fe storage. Cells transformed with the empty vector (pDRf1‐GW) or expressing SlVIT1, SlVIT2, SlVTL1 or SlVTL2 were spotted on SD without uracil, supplemented or not with 5 mM FeSO4. (b) Complementation assay of the pmr1Δ yeast mutant, which is sensitive to Mn toxicity. Cells expressing the same constructs were grown on SD medium lacking uracil, supplemented or not with 5 mM MnCl2. The images shown are representative of each condition from three plates, repeated in two independent experiments.

Given that the yeast vacuolar transporter ScCcc1 has also been reported to mediate manganese (Mn) transport to the vacuole or Golgi vesicles (Lapinskas et al. 1996), the ability of the SlVIT/VTL proteins to complement Mn sensitivity of the pmr1Δ yeast mutant was evaluated. This mutant cells are defective in Mn transport to the Golgi vesicles and are unable to grow under Mn toxic concentration (Lapinskas et al. 1995). All mutant yeast expressing SlVIT/VTL genes restored the ability of the mutant cells to grow in the Mn‐supplemented media, indicating that all proteins are capable of Mn transport (Figure 6b). Taken together, these results suggest that SlVIT1, SlVTL1 and SlVTL2 are functional as dual Fe and Mn transporters, whereas SlVIT2 appears to act as a Mn transporter.

3.6. Fe Deficiency and Mycorrhiza Shape the Tomato Ionome

Considering the known interconnections between Fe and various macro‐ and micronutrients as well as the role of AM in plant mineral nutrition, we analyzed the elemental composition of plants subjected to the different treatments. While most studies focus on mineral nutrient concentrations, we chose to analyze total nutrient contents to better understand differences in uptake and distribution. A supervised analysis (sPLSDA) revealed that both AM colonization and Fe deficiency significantly influenced the nutrient profiles of shoots and roots (Figure 7). In shoots, Fe availability had a greater impact on nutrient composition than AM colonization when compared with non‐mycorrhizal control plants. In contrast, in roots, the most pronounced effect was exerted by AM colonization, leading to a marked shift in nutrient distribution regardless of Fe status.

Figure 7.

Figure 7

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Effect of Fe deficiency and mycorrhizal colonization on the Solanum lycopersicum ionome. Nutrient profiles of shoots (a) and roots (b) from mycorrhizal (Myc) and non‐mycorrhizal (NM) plants grown with either half‐strength Hoagland solution containing 50 µM Fe (Control) or a modified nutrient solution lacking Fe (‐Fe). Left panels: Principal Component Analyses (PCA) showing sample clustering based on nutrient composition of three biological samples; each sample corresponds to a pool of two plants per treatment. Right panels: Heat maps and hierarchical clustering of nutrient contents across treatments. Data represent means of three biological replicates per treatment, each replicate consisting of a pool of two plants. [Color figure can be viewed at wileyonlinelibrary.com]

Iron deficiency significantly altered the shoot ionome; however, these changes were less pronounced in mycorrhizal plants. In non‐mycorrhizal plants, Fe deficiency significantly increased shoot contents of calcium, phosphorus and sulfur, while reducing the levels of Fe, copper, potassium, sodium, and zinc. In contrast, in mycorrhizal plants, shoot contents of calcium, sulfur, potassium, and zinc remained unaffected by Fe deficiency. Nevertheless, similar to non‐mycorrhizal plants, Fe deficiency still resulted in elevated phosphorus levels and reduced Fe, copper, and sodium contents in shoots (Supporting Information S2: Figures 2 and 8a). These patterns suggest that AM symbiosis partially mitigates shoot nutrient imbalances under low Fe conditions. In roots, Fe‐deficient conditions affected only the molybdenum content in mycorrhizal plants, without causing statistically significant changes in the concentrations of other nutrients (Supporting Information S2: Figures 3 and 8a).

Figure 8.

Figure 8

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Schematic summary of the effects of Fe availability and mycorrhizal colonization on plant nutrient profiles. (a) Nutrient shifts induced by Fe deficiency (−Fe) in non‐mycorrhizal (NM; upper panel) and mycorrhizal (Myc; lower panel) plants, highlighting which elements accumulate or decline under Fe limited supply and how AM partially mitigates these Fe‐deficiency‐driven ionomic changes. (b) Nutrient changes associated with AM colonization under Fe‐replete (+ Fe; upper panel) and Fe deficient conditions (−Fe; lower panel), illustrating how AM symbiosis reshapes the plant ionome according to Fe status. Green arrows indicate increases; red arrows indicate decreases. [Color figure can be viewed at wileyonlinelibrary.com]

To further assess the role of AM symbiosis in nutrient homeostasis, we compared ionomic profiles under Fe‐sufficient and Fe‐deficient conditions. Under Fe‐sufficiency, AM colonization significantly influenced shoot nutrient composition, with mycorrhizal plants exhibiting higher shoot sulfur levels and lower potassium and Fe contents compared to non‐mycorrhizal counterparts. Under Fe‐deficient conditions, however, no significant differences were observed in the shoot ionome between non‐mycorrhizal and mycorrhizal plants (Supporting Information S2: Figures 2 and 8b). In roots, AM colonization consistently increased copper and Fe contents, regardless of Fe availability. Zinc, manganese, molybdenum and phosphorus levels also tended to be higher in mycorrhizal roots, although increases in zinc, manganese and phosphorus were statistically significant only under Fe‐sufficient conditions, whereas molybdenum showed a significant increase under Fe‐deficient conditions (Supporting Information S2: Figures 3 and 8b).

4. Discussion

Despite the well‐established role of AM symbiosis in plant mineral nutrition, its contribution to Fe homeostasis remains insufficiently explored. This study provides compelling evidence that AM symbiosis modulates Fe homeostasis in tomato, integrating both physiological and molecular responses. Our findings demonstrate that the AM fungus R. irregularis alters Fe distribution and nutrient profiles in tomato and that Fe deficiency inhibits mycorrhization.

4.1. Plant Growth Responses to Fe Deficiency and the Contribution of AM Symbiosis

From a physiological perspective, the increase in root biomass under Fe deficiency in non‐mycorrhizal plants likely reflects an adaptive strategy to enhance soil exploration. In contrast, mycorrhizal plants did not exhibit this response, possibly due to the extended absorptive surface provided by the extraradical mycelium. While most studies report a reduction in plant biomass under Fe deficiency (Jin et al. 2009; Ravet et al. 2012; Jin et al. 2013; Takahashi et al. 2001), mild Fe deficiency has been shown to have limited impact on growth (Gruber et al. 2013). Considering that optimal plant growth requires Fe concentrations between 50 and 150 ppm (Marschner 2012), the Fe‐deficient treatment applied in our study likely represents a moderate deficiency. This is supported by the fact that shoot Fe concentration in plants grown without Fe supplementation reached 35 ppm. Such a moderate deficiency could explain why no significant differences were observed in shoot biomass between non‐mycorrhizal plants grown under Fe‐sufficient and Fe‐deficient conditions. This moderate deficiency may be attributed to the low P concentration (0.125 mM P) in the nutrient solution, which was intentionally reduced to avoid the inhibitory effects of high P levels on AM colonization. Antagonistic interactions between P and Fe deficiencies have been reported in several plant species (Hirsch et al. 2006; Müller et al. 2007; Thibaud et al. 2010; Yang et al. 2024).

4.2. Arbuscule‐Colonized Cells as Fe Sinks: Implications for Fe Retention

Our findings showing increased Fe content in mycorrhizal roots are consistent with a previous meta‐analysis on the role of AM symbiosis in crop micronutrient nutrition, which revealed that the positive effect of AM on Fe nutrition is primarily observed in roots (Lehmann and Rillig 2015). Notably, we report for the first time an accumulation of Fe in the cortical cells colonized by arbuscules. This observation suggests a local redistribution of Fe in arbuscule‐containing cells during the R. irregularis‐tomato symbiosis. One possible explanation is that the fungus functions as a sink for Fe, a hypothesis supported by previous findings showing high expression levels of the R. irregularis Fe uptake transporter RiFTR1 in mycorrhizal roots, particularly in arbuscule‐containing cells (Tamayo et al. 2018, 2025). At the same time, colonised cortical cells may retain Fe to support the profound structural and metabolic reprogramming associated with fungal colonization (Ferreras‐Garrucho et al. 2025). In this context, the observed reduction in the expression of the plant vacuolar Fe transporter genes SlVIT1 and SlVTL1 could be consistent with a decreased sequestration of Fe into vacuoles, thereby increasing cytosolic Fe availability in arbuscule‐containing cells. Together, these observations suggest that both fungal uptake and host cell retention could contribute to the observed Fe accumulation. A similar pattern of Fe distribution has been observed in the legume‐rhizobium symbiosis, where root nodules have been identified as major Fe sinks. In this symbiotic context, Fe is a critical micronutrient, serving as a cofactor of nitrogenase and various other enzymes essential for the nitrogen fixation process (Brear et al. 2020). The role of Fe in plant‐microbe interactions has long been recognized (Verbon et al. 2017). Iron can act as bargaining chip: microbes require it for infection and growth, while plants actively withhold it (Trapet et al. 2021). Plants have been known to employ strategies to extract Fe from pathogens (Eichhorn et al. 2006), but they must also supply it to beneficial microbes involved in symbiotic or endophytic associations (Brear et al. 2020; Johnson et al. 2013).

4.3. Iron Availability Regulates AM Symbiosis

Consistent with the importance of Fe in symbiotic interactions, the down‐regulation of the tomato AM‐related genes SlEXO84, SlRAM1, SlAMT2.2 and SlPT4, and the lower RiEF1α expression in mycorrhizal roots under Fe‐deficient conditions, indicates that Fe availability is crucial for AM colonization and functionality in tomato. Given that Fe is essential for both plant and fungal metabolism, the inhibition of mycorrhizal colonization under Fe deficiency likely reflects the critical role of this micronutrient in AM establishment and function. Supporting this, overexpression of the R. irregularis Fe transporter RiFTR1 in Medicago truncatula roots has been shown to promote arbuscule development and symbiotic phosphate transport (Tamayo et al. 2025). Similarly, Fe deficiency has been reported to inhibit nodule development and nitrogen fixation (Chu et al. 2019), and to limit infection by pathogens with diverse lifestyles, from necrotrophs to biotrophs (Trapet et al. 2021). The inhibition of AM colonization under Fe‐deficient conditions may also be linked to increased phosphorus accumulation in mycorrhizal shoots, as high phosphorus levels are known to supress AM development (Shi et al. 2021).

4.4. AM Symbiosis Modulates Fe Uptake Pathways in an Fe‐Dependent Manner

Regarding the impact of AM on the expression of Fe‐responsive genes of tomato, our data reveal an Fe‐dependent differential regulation of key genes involved in Fe uptake, distribution and storage. Under Fe‐limiting conditions, SlFRO1 and SlIRT1 are up‐regulated in mycorrhizal roots, supporting the view of increased Fe bioavailability in the rhizosphere of mycorrhizal plants (Kabir et al. 2020; Rahimi et al. 2021). Since SlFRO1 encodes the ferric reductase required for Strategy I Fe uptake (Robinson et al. 1999), its induction together with that of SlIRT1 likely contributes to the higher Fe content observed in mycorrhizal roots. In contrast, under Fe sufficient conditions, SlFRO1 is undetectable and SlIRT1 is down‐regulated in mycorrhizal roots, a pattern also reported in alfalfa, quince and sunflower roots (Kabir et al. 2020; Rahman et al. 2020; Rahimi et al. 2021). These findings indicate that AM enhances the Strategy I Fe acquisition pathway only when Fe is limiting, highlighting the context‐dependent nature of AM‐mediated regulation of Fe homeostasis. The down‐regulation of SlIRT1 under optimal conditions suggests that direct epidermal Fe uptake is reduced in mycorrhizal roots, similar to the down‐regulation of the direct uptake pathways described for phosphorus, nitrogen and zinc (Coccina et al. 2019; Liu et al. 1998; Pérez‐Tienda et al. 2014). Interestingly, increased transcript levels of SlNRAMP1 in mycorrhizal roots under optimal Fe conditions suggest that its encoded protein may participate in Fe uptake via the mycorrhizal pathway, potentially explaining the increased Fe content in these roots. Although functional characterization is still pending, SlNRAMP1 appears to be a promising candidate for mediating Fe transport from fungal structures to the plant.

The down‐regulation of SlCHLN, involved in nicotianamine biosynthesis, under Fe‐deficient conditions and in mycorrhizal roots, agrees with the observed decrease in root‐to‐shoot Fe translocation. Given the essential role of nicotianamine in long‐distance Fe transport (Higuchi et al. 1996), reduced biosynthesis could limit Fe mobility and favor its retention in the root. This idea aligns with previous findings in Arabidopsis showing that nicotianamine overaccumulation enhances Fe translocation to the shoot and reduces its retention in the roots (Haydon et al. 2012). Thus, the lower expression of SlCHLN in mycorrhizal and Fe‐deficient roots likely reflects a physiological adjustment that prioritizes local Fe retention over systemic distribution, possibly to support root‐associated symbiotic functions or to cope with limited Fe availability. Assessing nicotianamine levels and Fe–nicotianamine complexes will be important to validate this model and further understand the regulatory links between Fe homeostasis and AM symbiosis.

4.5. Functional Roles of SlVIT/VTL Transporters and Their Regulation by AM

The high demand of Fe for AM was supported by the reduced expression of the vacuolar Fe transporter SlVIT1 and SlVTL1 in mycorrhizal roots. Our functional analyses revealed that SlVIT1, SlVTL1 and SlVTL2 are capable of transporting both Fe and manganese, while SlVIT2 appears to be a Mn transporter. Consistent with their role in Fe homeostasis by sequestering Fe into the vacuole, the expression of SlVIT1, SlVTL1 and SlVTL2 was down‐regulated under Fe‐deficient conditions. Interestingly, SlVIT2 expression was up‐regulated in mycorrhizal roots, accompanied by increased Mn, suggesting an active role of this micronutrient in AM. Increased Mn tissue concentrations in mycorrhizal plants have been reported in several plant species (Lambert et al. 1979; Eivazi and Weir 1989). The biological relevance of this Mn enrichment may lie in the diverse roles this micronutrient plays in plant physiology, including photosynthesis, respiration, scavenging of reactive oxygen species, pathogen defence and hormone signalling (Alejandro et al. 2020). Further research is needed to clarify the functional significance of SlVIT2 in AM symbiosis.

4.6. AM Symbiosis Buffers Nutrient Imbalances Under Fe Deficiency

Regarding the ionomic data, our results indicate that AM colonization partially mitigates the alterations induced in the tomato shoot ionome under Fe‐deficient conditions. Iron deficiency is known to exert both direct and indirect effects on nutrient uptake, distribution, and metabolic homeostasis, leading to substantial changes in the plant ionome. One of the most notable adaptive responses to Fe deficiency involves the modulation of sulphate uptake and assimilation (Astolfi et al. 2010; Ciaffi et al. 2013; Paolacci et al. 2014), as Fe shortage increases sulphur demand and activates pathways similar to those triggered by sulphate‐deficient conditions. Likewise, Fe deficiency has been shown to induce phosphate accumulation through the up‐regulation of phosphate‐related genes (Moran Lauter et al. 2014). Additionally, cross‐talk between calcium, phosphorus and Fe has been reported (Matthus et al. 2019), further highlighting the complexity of nutrient interactions under Fe‐limited conditions. Micronutrient levels are also affected: manganese tends to accumulate due to reduced transporter selectivity, while zinc and copper levels may decline, depending on species and soil characteristics. These shifts underscore the intricate interplay among mineral elements and the broader impact of Fe deficiency on plant nutritional balance. Our findings, showing that calcium, sulphur, potassium and zinc levels remained unaffected by Fe deficiency in AM‐colonized plants, underscore the role of AM symbiosis in buffering nutrient imbalances and maintaining a more stable ionomic profile under Fe stress. The mechanisms underlying this buffering effect—likely encompassing physiological, molecular, and multi‐elemental uptake processes—remain to be elucidated. Our data also show that the impact of AM symbiosis on nutrient profiles is highly dependent on Fe availability, exerting a pronounced influence under Fe sufficiency but a more limited effect during Fe deficiency. This context‐dependent response suggests that Fe availability constrains the extent to which AM fungi can modify host nutrient profiles.

5. Conclusion

The data presented in this manuscript demonstrate that Fe availability is a key determinant of AM symbiosis in tomato. AM colonization modulates the expression of Fe‐related genes involved in uptake, transport and storage, while Fe accumulated in arbuscule‐containing cells, suggesting that both the fungus and the colonized host cells could act as a Fe sinks. Additionally, increased Mn levels and SlVIT2 induction in AM roots point to a broader role of micronutrient homeostasis in symbiosis. Together, these findings reveal a complex, context‐dependent regulation of nutrient pathways that balances plant and fungal needs, and underscore the importance of further exploring the mechanisms of Fe transfer and micronutrient dynamics in AM interactions.

Supporting information

Supplementary Figure 1: (a) Predicted 3D structure of S. lycopersicum VIT/VTL protein monomers. Supplementary Figure 2: Shoot nutrient content of tomato non‐mycorrhizal (NM) and mycorrhizal (Myc) plants fertilized with half‐strength Hoagland solution (Control, 50 μM Fe) or a modified nutrient solution without Fe (‐Fe). Supplementary Figure 3: Root nutrient content of tomato non‐mycorrhizal (NM) and mycorrhizal (Myc) plants fertilized with half‐strength Hoagland solution (Control, 50 μM Fe) or a modified nutrient solution without Fe (‐Fe).

PCE-49-3290-s002.pdf (541.5KB, pdf)

Supplementary Table 1: Oligonucleotides used in this study. Overhangs are underlined. Supplementary Table 2: SlVIT/VTL protein accession numbers.

PCE-49-3290-s001.docx (29.4KB, docx)

Acknowledgements

This work was supported by grant PID2021‐1255210B‐I00 funded by MCIN/AEI/https://doi.org/10.13039/501100011033 and by “ERDF A way of making Europe”, by the “European Union”. Víctor Manuel López‐Lorca was supported by grant BES‐2016‐078463 funded by MCIN/AEI/https://doi.org/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Alejandro, S. , Höller S., Meier B., and Peiter E.. 2020. “Manganese in Plants: From Acquisition to Subcellular Allocation.” Frontiers in Plant Science 11: 300. 10.3389/fpls.2020.00300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Astolfi, S. , Zuchi S., Hubberten H. M., Pinton R., and Hoefgen R.. 2010. “Supply of Sulphur to S‐Deficient Young Barley Seedlings Restores Their Capability to Cope With Iron Shortage.” Journal of Experimental Botany 61: 799–806. 10.1093/jxb/erp346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Azcón‐Aguilar, C. , and Barea J. M.. 2015. “Nutrient Cycling in the Mycorrhizosphere.” Journal of Soil Science and Plant Nutrition 15: 372–396. 10.4067/S0718-95162015005000035. [DOI] [Google Scholar]
  4. Bereczky, Z. , Wang H.‐Y., Schubert V., Ganal M., and Bauer P.. 2003. “Differential Regulation of nramp and irt Metal Transporter Genes in Wild Type and Iron Uptake Mutants of Tomato.” Journal of Biological Chemistry 278: 24697–24704. 10.1074/jbc.M301365200. [DOI] [PubMed] [Google Scholar]
  5. Brear, E. M. , Bedon F., Gavrin A., et al. 2020. “GmVTL1a Is an Iron Transporter on the Symbiosome Membrane of Soybean With an Important Role in Nitrogen Fixation.” New Phytologist 228: 667–681. 10.1111/nph.16734. [DOI] [PubMed] [Google Scholar]
  6. Caris, C. , Hördt W., Hawkins H.‐J., Römheld V., and George E.. 1998. “Studies of Iron Transport by Arbuscular Mycorrhizal Hyphae From Soil to Peanut and Sorghum Plants.” Mycorrhiza 8: 35–39. 10.1007/s005720050208. [DOI] [Google Scholar]
  7. Chu, Q. , Sha Z., Maruyama H., et al. 2019. “Metabolic Reprogramming in Nodules, Roots, and Leaves of Symbiotic Soybean in Response to Iron Deficiency.” Plant Cell & Environment 42: 3027–3043. [DOI] [PubMed] [Google Scholar]
  8. Ciaffi, M. , Paolacci A. R., Celletti S., Catarcione G., Kopriva S., and Astolfi S.. 2013. “Transcriptional and Physiological Changes in the S Assimilation Pathway Due to Single or Combined S and Fe Deprivation in Durum Wheat (Triticum durum L.) Seedlings.” Journal of Experimental Botany 64: 1663–1675. 10.1093/jxb/ert027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clark, R. B. , and Zeto S. K.. 2000. “Mineral Acquisition by Arbuscular Mycorrhizal Plants.” Journal of Plant Nutrition 23: 867–902. 10.1080/01904160009382068. [DOI] [Google Scholar]
  10. Coccina, A. , Cavagnaro T. R., Pellegrino E., Ercoli L., McLaughlin M. J., and Watts‐Williams S. J.. 2019. “The Mycorrhizal Pathway of Zinc Uptake Contributes to Zinc Accumulation in Barley and Wheat Grain.” BMC Plant Biology 19: 133. 10.1186/s12870-019-1741-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Connorton, J. M. , Balk J., and Rodríguez‐Celma J.. 2017. “Iron Homeostasis in Plants—A Brief Overview.” Metallomics: Integrated Biometal Science 9: 813–823. 10.1039/c7mt00136c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duan, S. , Feng G., Limpens E., Bonfante P., Xie X., and Zhang L.. 2024. “Cross‐Kingdom Nutrient Exchange in the Plant–Arbuscular Mycorrhizal Fungus–Bacterium Continuum.” Nature Reviews Microbiology 22: 773–790. 10.1038/s41579-024-01073-7. [DOI] [PubMed] [Google Scholar]
  13. Eichhorn, H. , Lessing F., Winterberg B., et al. 2006. “A Ferroxidation/Permeation Iron Uptake System Is Required for Virulence in Ustilago maydis .” Plant Cell 18: 3332–3345. 10.1105/tpc.106.043588. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eivazi, F. , and Weir C. C.. 1989. “Phosphorus and Mycorrhizal Interaction on Uptake of P and Trace Elements by Maize.” Fertilizer Research 21: 19–22. [Google Scholar]
  15. Fan, X. , Zhou X., Chen H., Tang M., and Xie X.. 2021. “Cross‐Talks Between Macro‐ and Micronutrient Uptake and Signaling in Plants.” Frontiers in Plant Science 12: 663477. 10.3389/fpls.2021.663477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ferreras‐Garrucho, G. , Chancellor T., and Paszkowsk U.. 2025. “Integrating Single‐Cell Omic Techniques to Resolve the Spatio‐Temporal Complexity of Arbuscular Mycorrhizal Symbiosis.” Journal of Experimental Botany: 1–18. 10.1093/jxb/eraf404. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ferrol, N. , Tamayo E., and Vargas P.. 2016. “The Heavy Metal Paradox in Arbuscular Mycorrhizas: From Mechanisms to Biotechnological Applications.” Journal of Experimental Botany 67: 6253–6265. 10.1093/jxb/erw403. [DOI] [PubMed] [Google Scholar]
  18. García‐Rodríguez, S. , Azcón‐Aguilar C., and Ferrol N.. 2007. “Transcriptional Regulation of Host Enzymes Involved in the Cleavage of Sucrose During Arbuscular Mycorrhizal Symbiosis.” Physiologia Plantarum 129: 737–746. 10.1111/j.1399-3054.2007.00873.x. [DOI] [Google Scholar]
  19. Grotz, N. , and Guerinot M. L.. 2006. “Molecular Aspects of Cu, Fe and Zn Homeostasis in Plants.” Biochimica et Biophysica Acta (BBA)— Molecular Cell Research 1763: 595–608. 10.1016/j.bbamcr.2006.05.014. [DOI] [PubMed] [Google Scholar]
  20. Gruber, B. D. , Giehl R. F. H., Friedel S., and von Wirén N.. 2013. “Plasticity of the Arabidopsis Root System Under Nutrient Deficiencies.” Plant Physiology 163: 161–179. 10.1104/pp.113.218453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Haydon, M. J. , Kawachi M., Wirtz M., Hillmer S., Hell R., and Krämer U.. 2012. “Vacuolar Nicotianamine Has Critical and Distinct Roles Under Iron Deficiency and for Zinc Sequestration in Arabidopsis .” Plant Cell 24: 724–737. 10.1105/tpc.111.095042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Higuchi, K. , Nishizawa N., Römheld V., Marschner H., and Mori S.. 1996. “Absence of Nicotianamine Synthase Activity in the Tomato Mutant chloronerva .” Journal of Plant Nutrition 19: 1235–1239. 10.1080/01904169609365194. [DOI] [Google Scholar]
  23. Hirsch, J. , Marin E., Floriani M., et al. 2006. “Phosphate Deficiency Promotes Modification of Iron Distribution in Arabidopsis Plants.” Biochimie 88: 1767–1771. 10.1016/j.biochi.2006.05.007. [DOI] [PubMed] [Google Scholar]
  24. Ho‐Plágaro, T. , Huertas R., Tamayo‐Navarrete M. I., Ocampo J. A., and García‐Garrido J. M.. 2018. “An Improved Method for Agrobacterium rhizogenes‐Mediated Transformation of Tomato Suitable for the Study of Arbuscular Mycorrhizal Symbiosis.” Plant Methods 14: 34. 10.1186/s13007-018-0304-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ho‐Plágaro, T. , Morcillo R. J. L., Tamayo‐Navarrete M. I., et al. 2021. “DLK2 Regulates Arbuscule Hyphal Branching During Arbuscular Mycorrhizal Symbiosis.” New Phytologist 229: 548–562. 10.1111/nph.16938. [DOI] [PubMed] [Google Scholar]
  26. Ivanov, R. , Brumbarova T., Blum A., Jantke A. M., Fink‐Straube C., and Bauer P.. 2014. “Sorting NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis Iron‐Regulated Transporter1.” Plant Cell 26: 1294–1307. 10.1105/tpc.113.116244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jin, C. W. , Du S. T., Chen W. W., Li G. X., Zhang Y. S., and Zheng S. J.. 2009. “Elevated Carbon Dioxide Improves Plant Iron Nutrition Through Enhancing the Iron‐Deficiency‐Induced Responses Under Iron‐Limited Conditions in Tomato.” Plant Physiology 150: 272–280. 10.1104/pp.109.136721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jin, C.‐W. , Liu Y., Mao Q.‐Q., Wang Q., and Du S.‐T.. 2013. “Mild Fe‐Deficiency Improves Biomass Production and Quality of Hydroponic‐Cultivated Spinach Plants (Spinacia oleracea L.).” Food Chemistry 138: 2188–2194. 10.1016/j.foodchem.2012.12.025. [DOI] [PubMed] [Google Scholar]
  29. Johnson, L. J. , Koulman A., Christensen M., et al. 2013. “An Extracellular Siderophore Is Required to Maintain the Mutualistic Interaction of Epichloë festucae With Lolium perenne .” PLoS Pathogens 9: e1003332. 10.1371/journal.ppat.1003332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kabir, A. H. , Debnath T., Das U., et al. 2020. “Arbuscular Mycorrhizal Fungi Alleviate Fe‐Deficiency Symptoms in Sunflower by Increasing Iron Uptake and Its Availability Along With Antioxidant Defense.” Plant Physiology and Biochemistry 150: 254–262. 10.1016/j.plaphy.2020.03.010. [DOI] [PubMed] [Google Scholar]
  31. Kabir, A. H. , Paltridge N. G., Roessner U., and Stangoulis J. C. R.. 2013. “Mechanisms Associated With Fe‐Deficiency Tolerance and Signaling in Shoots of Pisum sativum .” Physiologia Plantarum 147: 381–395. 10.1111/j.1399-3054.2012.01682.x. [DOI] [PubMed] [Google Scholar]
  32. Kato, T. , Kumazaki K., Wada M., et al. 2019. “Crystal Structure of Plant Vacuolar Iron Transporter VIT1.” Nature Plants 5: 308–315. 10.1038/s41477-019-0367-2. [DOI] [PubMed] [Google Scholar]
  33. Kobae, Y. , Tomioka R., Tanoi K., et al. 2014. “Selective Induction of Putative Iron Transporters, OPT8a and OPT8b, in Maize by Mycorrhizal Colonization.” Soil Science and Plant Nutrition 60: 843–847. 10.1080/00380768.2014.949854. [DOI] [Google Scholar]
  34. Lambert, D. H. , Baker D. E., and Cole H. Jr. 1979. “The Role of Mycorrhizae in the Interactions of Phosphorus With Zinc, Copper, and Other Elements.” Soil Science Society of America Journal 43: 976–980. 10.2136/sssaj1979.03615995004300050033x. [DOI] [Google Scholar]
  35. Lapinskas, P. J. , Cunningham K. W., Liu X. F., Fink G. R., and Culotta V. C.. 1995. “Mutations in PMR1 Suppress Oxidative Damage in Yeast Cells Lacking Superoxide Dismutase.” Molecular and Cellular Biology 15: 1382–1388. 10.1128/MCB.15.3.1382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lapinskas, P. J. , Lin S.‐J., and Culotta V. C.. 1996. “The Role of the Saccharomyces cerevisiae CCC1 Gene in the Homeostasis of Manganese Ions.” Molecular Microbiology 21: 519–528. 10.1111/j.1365-2958.1996.tb02561.x. [DOI] [PubMed] [Google Scholar]
  37. Lehmann, A. , and Rillig M. C.. 2015. “Arbuscular Mycorrhizal Contribution to Copper, Manganese and Iron Nutrient Concentrations in Crops—A Meta‐Analysis.” Soil Biology and Biochemistry 81: 147–158. 10.1016/j.soilbio.2014.11.013. [DOI] [Google Scholar]
  38. Li, L. , Chen O. S., Ward D. M., and Kaplan J.. 2001. “CCC1 Is a Transporter That Mediates Vacuolar Iron Storage in Yeast.” Journal of Biological Chemistry 276: 29515–29519. 10.1074/jbc.M103944200. [DOI] [PubMed] [Google Scholar]
  39. Liang, G. 2022. “Iron Uptake, Signaling, and Sensing in Plants.” Plant Communications 3: 100349. 10.1016/j.xplc.2022.100349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Liu, H. , Trieu A. T., Blaylock L. A., and Harrison M. J.. 1998. “Cloning and Characterization of Two Phosphate Transporters From Medicago truncatula Roots: Regulation in Response to Phosphate and to Colonization by Arbuscular Mycorrhizal (AM) Fungi.” Molecular Plant‐Microbe Interactions® 11: 14–22. 10.1094/MPMI.1998.11.1.14. [DOI] [PubMed] [Google Scholar]
  41. Liu, Y. , Xiong Z., Wu W., Ling H.‐Q., and Kong D.. 2023. “Iron in the Symbiosis of Plants and Microorganisms.” Plants 12: 1958. 10.3390/plants12101958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lurthy, T. , Cantat C., Jeudy C., et al. 2020. “Impact of Bacterial Siderophores on Iron Status and Ionome in Pea.” Frontiers in Plant Science 11: 730. 10.3389/fpls.2020.00730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Lurthy, T. , Pivato B., Lemanceau P., and Mazurier S.. 2021. “Importance of the Rhizosphere Microbiota in Iron Biofortification of Plants.” Frontiers in Plant Science 12: 744445. 10.3389/fpls.2021.744445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Marschner, P. 2012. Marschner's Mineral Nutrition of Higher Plants (Third). Academic Press.
  45. Marschner, P. , Crowley D., and Rengel Z.. 2011. “Rhizosphere Interactions Between Microorganisms and Plants Govern Iron and Phosphorus Acquisition Along the Root Axis—Model and Research Methods.” Soil Biology and Biochemistry 43: 883–894. 10.1016/j.soilbio.2011.01.005. [DOI] [Google Scholar]
  46. Matthus, E. , Wilkins K. A., and Davies J. M.. 2019. “Iron Availability Modulates the Arabidopsis thaliana Root Calcium Signature Evoked by Exogenous ATP.” Plant Signaling & Behavior 14: 1640563. 10.1080/15592324.2019.1640563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Moran Lauter, A. N. , Peiffer G. A., Yin T., and Moose S. P.. 2014. “Iron Deficiency in Maize Induces Transcriptome Changes in Genes Involved in Iron Homeostasis and Phosphorus Metabolism.” Plant Physiology 165: 743–756. 10.1104/pp.114.238998. [DOI] [Google Scholar]
  48. Moreno Jiménez, E. , Ferrol N., Corradi N., Peñalosa J. M., and Rillig M. C.. 2024. “The Potential of Arbuscular Mycorrhizal Fungi to Enhance Metallic Micronutrient Uptake and Mitigate Food Contamination in Agriculture: Prospects and Challenges.” New Phytologist 242: 1441–1447. 10.1111/nph.19269. [DOI] [PubMed] [Google Scholar]
  49. Müller, R. , Morant M., Jarmer H., Nilsson L., and Nielsen T. H.. 2007. “Genome‐Wide Analysis of the Arabidopsis Leaf Transcriptome Reveals Interaction of Phosphate and Sugar Metabolism.” Plant Physiology 143: 156–171. 10.1104/pp.106.090167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Paolacci, A. R. , Celletti S., Catarcione G., Hawkesford M. J., Astolfi S., and Ciaffi M.. 2014. “Iron Deprivation Results in a Rapid but Not Sustained Increase of the Expression of Genes Involved in Iron Metabolism and Sulfate Uptake in Tomato (Solanum lycopersicum L.) Seedlings.” Journal of Integrative Plant Biology 56: 88–100. 10.1111/jipb.12116. [DOI] [PubMed] [Google Scholar]
  51. Pérez‐Tienda, J. , Corrêa A., Azcón‐Aguilar C., and Ferrol N.. 2014. “Transcriptional Regulation of Host Transporters and GS/GOGAT Pathway in Arbuscular Mycorrhizal Rice Roots.” Plant Physiology and Biochemistry 75: 1–8. 10.1016/j.plaphy.2013.11.029. [DOI] [PubMed] [Google Scholar]
  52. Prity, S. A. , Sajib S. A., Das U., Rahman M. M., Haider S. A., and Kabir A. H.. 2020. “Arbuscular Mycorrhizal Fungi Mitigate Fe Deficiency Symptoms in Sorghum Through Phytosiderophore‐Mediated Fe Mobilization and Restoration of Redox Status.” Protoplasma 257: 1373–1385. 10.1007/s00709-020-01517-w. [DOI] [PubMed] [Google Scholar]
  53. Rahimi, S. , Baninasab B., Talebi M., Gholami M., and Zarei M.. 2021. “Arbuscular Mycorrhizal Fungi Inoculation Improves Iron Deficiency in Quince via Alterations in Host Root Phenolic Compounds and Expression of Genes.” Scientia Horticulturae 285: 110165. 10.1016/j.scienta.2021.110165. [DOI] [Google Scholar]
  54. Rahman, M. A. , Parvin M., Das U., et al. 2020. “Arbuscular Mycorrhizal Symbiosis Mitigates Iron (Fe)‐Deficiency Retardation in Alfalfa (Medicago sativa L.) Through the Enhancement of Fe Accumulation and Sulfur‐Assisted Antioxidant Defense.” International Journal of Molecular Sciences 21: 2219. 10.3390/ijms21062219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Rai, S. , Singh P. K., Mankotia S., Swain J., and Satbhai S. B.. 2021. “Iron Homeostasis in Plants and Its Crosstalk With Copper, Zinc, and Manganese.” Plant Stress 1: 100008. 10.1016/j.stress.2021.100008. [DOI] [Google Scholar]
  56. Rajapitamahuni, S. , Kang B. R., and Lee T. K.. 2023. “Exploring the Roles of Arbuscular Mycorrhizal Fungi in Plant–Iron Homeostasis.” Agriculture (London) 13: 1918. 10.3390/agriculture13101918. [DOI] [Google Scholar]
  57. Ram, H. , Sardar S., and Gandass N.. 2021. “Vacuolar Iron Transporter (Like) Proteins: Regulators of Cellular Iron Accumulation in Plants.” Physiologia Plantarum 171: 823–832. 10.1111/ppl.13363. [DOI] [PubMed] [Google Scholar]
  58. Ravet, K. , Reyt G., Arnaud N., et al. 2012. “Iron and ROS Control of the DownSTream mRNA Decay Pathway Is Essential for Plant Fitness.” EMBO Journal 31: 175–186. 10.1038/emboj.2011.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Robinson, N. J. , Procter C. M., Connolly E. L., and Guerinot M. L.. 1999. “A Ferric‐Chelate Reductase for Iron Uptake From Soils.” Nature 397: 694–697. 10.1038/17800. [DOI] [PubMed] [Google Scholar]
  60. Schiestl, R. H. , and Gietz R. D.. 1989. “High Efficiency Transformation of Intact Yeast Cells Using Single Stranded Nucleic Acids as a Carrier.” Current Genetics 16: 339–346. 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  61. Schmittgen, T. D. , and Livak K. J.. 2008. “Analyzing Real‐Time PCR Data by the Comparative CT Method.” Nature Protocols 3: 1101–1108. 10.1038/nprot.2008.73. [DOI] [PubMed] [Google Scholar]
  62. Shi, J. , Zhao B., Zheng S., et al. 2021. “A Phosphate Starvation Response‐Centered Network Regulates Mycorrhizal Symbiosis.” Cell 184: 5527–5540. [DOI] [PubMed] [Google Scholar]
  63. Smith, S. E. , and Read D. J.. 2008. Mycorrhizal Symbiosis. 3th ed. Academic Press. [Google Scholar]
  64. Sorribes‐Dauden, R. , Peris D., Martínez‐Pastor M. T., and Puig S.. 2020. “Structure and Function of the Vacuolar Ccc1/VIT1 Family of Iron Transporters and Its Regulation in Fungi.” Computational and Structural Biotechnology Journal 18: 3712–3722. 10.1016/j.csbj.2020.10.044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Suzuki, H. , Kumagai H., Oohashi K., et al. 2000. “Uptake of 15 Trace Elements in Arbuscular Mycorrhizal Marigold Measured by the Multitracer Technique.” Soil Science and Plant Nutrition 46: 283–289. 10.1080/00380768.2000.10408784. [DOI] [Google Scholar]
  66. Takahashi, M. , Nakanishi H., Kawasaki S., Nishizawa N. K., and Mori S.. 2001. “Enhanced Tolerance of Rice to Low Iron Availability in Alkaline Soils Using Barley Nicotianamine Aminotransferase Genes.” Nature Biotechnology 19: 466–469. 10.1038/88143. [DOI] [PubMed] [Google Scholar]
  67. Tamayo, E. , Knight S. A. B., Valderas A., Dancis A., and Ferrol N.. 2018. “The Arbuscular Mycorrhizal Fungus Rhizophagus irregularis Uses a Reductive Iron Assimilation Pathway for High‐Affinity Iron Uptake.” Environmental Microbiology 20: 1857–1872. 10.1111/1462-2920.14121. [DOI] [PubMed] [Google Scholar]
  68. Tamayo, E. , López‐Lorca V. M., Shim C., et al. 2025. “The Rhizophagus irregularis Permease RiFTR1 Functions Without a Ferroxidase Partner for Reductive Iron Transport.” Scientific Reports 15: 5840. 10.1038/s41598-025-88416-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Thibaud, M.‐C. , Arrighi J.‐F., Bayle V., et al. 2010. “Dissection of Local and Systemic Transcriptional Responses to Phosphate Starvation in Arabidopsis .” Plant Journal 64: 775–789. 10.1111/j.1365-313X.2010.04375.x. [DOI] [PubMed] [Google Scholar]
  70. Trapet, P. L. , Verbon E. H., Bosma R. R., Voordendag K., van Pelt J. A., and Pieterse C. M. J.. 2021. “Mechanisms Underlying Iron Deficiency‐Induced Resistance Against Pathogens With Different Lifestyles.” Journal of Experimental Botany 72: 2231–2241. 10.1093/jxb/eraa535. [DOI] [PubMed] [Google Scholar]
  71. Verbon, E. H. , Trapet P. L., Stringlis I. A., Kruijs S., Bakker P. A. H. M., and Pieterse C. M. J.. 2017. “Iron and Immunity.” Annual Review of Phytopathology 55: 355–375. 10.1146/annurev-phyto-080516-035537. [DOI] [PubMed] [Google Scholar]
  72. Yang, X. , Liu C., Liang C., Wang T., and Tian J.. 2024. “The Phosphorus‐Iron Nexus: Decoding the Nutrients Interaction in Soil and Plant.” International Journal of Molecular Sciences 25: 6992. 10.3390/ijms25136992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  73. Zamboni, A. , Zanin L., Tomasi N., et al. 2012. “Genome‐Wide Microarray Analysis of Tomato Roots Showed Defined Responses to Iron Deficiency.” BMC Genomics 13: 101. 10.1186/1471-2164-13-101. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Figure 1: (a) Predicted 3D structure of S. lycopersicum VIT/VTL protein monomers. Supplementary Figure 2: Shoot nutrient content of tomato non‐mycorrhizal (NM) and mycorrhizal (Myc) plants fertilized with half‐strength Hoagland solution (Control, 50 μM Fe) or a modified nutrient solution without Fe (‐Fe). Supplementary Figure 3: Root nutrient content of tomato non‐mycorrhizal (NM) and mycorrhizal (Myc) plants fertilized with half‐strength Hoagland solution (Control, 50 μM Fe) or a modified nutrient solution without Fe (‐Fe).

PCE-49-3290-s002.pdf (541.5KB, pdf)

Supplementary Table 1: Oligonucleotides used in this study. Overhangs are underlined. Supplementary Table 2: SlVIT/VTL protein accession numbers.

PCE-49-3290-s001.docx (29.4KB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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