Iron Availability Modulates Arbuscular Mycorrhizal Symbiosis, Which in Turn Influences the Expression of Iron-Responsive Genes and Nutrient Balance in Solanum lycopersicum

Iron Availability Modulates Arbuscular Mycorrhizal Symbiosis, Which in Turn Influences the Expression of Iron-Responsive Genes and Nutrient Balance in Solanum lycopersicum | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Plant, Cell & Environment This is a preprint and has not been peer reviewed. Data may be preliminary. 8 September 2025 V1 Latest version Share on Iron Availability Modulates Arbuscular Mycorrhizal Symbiosis, Which in Turn Influences the Expression of Iron-Responsive Genes and Nutrient Balance in Solanum lycopersicum Authors : Víctor M. López-Lorca , Olga López-Castillo , Mª Jesús Molina-Luzón , and Nuria Ferrol 0000-0001-7963-3537 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.175732766.62550850/v1 282 views 149 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share 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, suggesting that in the Rhizophagus irregularis -tomato symbiosis, the fungus acts as a sink for Fe. 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. The high Fe demand of AM symbiosis is supported by the reduced expression of the vacuolar Fe transporters SlVIT1 and SlVTL1 in mycorrhizal roots. Ionomic analysis reveals that AM colonization partially mitigates the alterations induced by Fe deficiency, underscoring the role of AM in buffering nutrient imbalances and maintaining a more stable ionomic profile under Fe stress. Iron Availability Modulates Arbuscular Mycorrhizal Symbiosis, Which in Turn Influences the Expression of Iron-Responsive Genes and Nutrient Balance in Solanum lycopersicum Víctor M. López-Lorca, Olga López-Castillo, Mª Jesús Molina-Luzón, Nuria Ferrol Soil and Plant Microbiology Department, Estación Experimental del Zaidín, CSIC, C. Profesor Albareda 1, 18008, Granada, Spain Corresponding author: Nuria Ferrol; [email protected] 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, suggesting that in the Rhizophagus irregularis -tomato symbiosis, the fungus acts as a sink for Fe. 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. The high Fe demand of AM symbiosis is supported by the reduced expression of the vacuolar Fe transporters SlVIT1 and SlVTL1 in mycorrhizal roots. Ionomic analysis reveals that AM colonization partially mitigates the alterations induced by Fe deficiency, underscoring the role of AM in buffering nutrient imbalances and maintaining a more stable ionomic profile under Fe stress. 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 a mechanism known as Strategy I, which involves a plasma membrane H + -ATPase that acidifies the rhizosphere, thereby solubilizing Fe chelates. This is followed the action of a ferric reductase (FRO1), which reduces Fe (III) to Fe (II), and the uptake of Fe (II) through specific transporters (IRT and NRAMP1). In contrast, grasses employ a Strategy II, which consists of the biosynthesis and release of phytosiderophores (PS) to the rhizosphere. These compounds chelate Fe (III), and the resulting PS-Fe (III) complexes are then transported into root cells via plasma membrane oligopeptide transporters of the YS1 or YS1-like family (Liang 2022). After uptake from the rhizosphere into the root epidermis, small molecules facilitate the solubilization and transport of Fe (Clemens 2019). Within the symplast, Fe is transported toward the endodermis primarily in the form of Fe (II)-nicotianamine complexes. Nicotianamine is a non-protein amino acid produced from S-adenosyl methionine by nicotianamine synthase. 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). In addition to these intrinsic Fe uptake mechanisms, plants have evolved alternative strategies to cope with Fe deficiency, including the establishment of associations with beneficial soil microorganisms (Marschner et al. 2011; Lurthy et al. 2021). One well-documented example is Fe acquisition mediated by siderophores produced by rhizosphere microorganisms, which can significantly contribute to plant Fe nutrition (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, which belong to the subphylum Glomeromycotina, establish a mutualistic symbiosis, known as arbuscular mycorrhiza, with the majority of terrestrial plant species (Azcón-Aguilar and Barea 2015). These fungi colonize the root cortex and develop an extensive network of extraradical hyphae in the surrounding soil. This external mycelium enables the uptake of nutrients beyond the root depletion zones, which are subsequently delivered to the plant through colonized cortical cells, where the fungus forms highly branched structures called arbuscules. In exchange, the plant supplies the fungus with carbon compounds derived from photosynthesis (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 et al. 2024). The role of AM fungi in plant Zn and Cu nutrition is well established. In contrast, the impact of mycorrhizal colonization on Fe nutrition remains variable and, in many cases, inconsistent (Clark and Zeto 2000). A meta-analysis of more of 200 studies supports a generally positive effect of AM fungi on Fe nutrition in crop plants (Lehman and Rillig 2015). However, the magnitude and direction of this effect depend on several factors, including plant growth conditions, host plant species, and the specific AM fungal taxa (Caris et al. 1998; Suzuki et al. 2000; Kobae et al. 2014). Some 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). These findings suggest that increased Fe uptake under Fe-limiting conditions in mycorrhizal plants may be linked to enhanced Fe bioavailability in the rhizosphere. This is supported by observations of increased phytosiderophore release in Strategy II plants (Prity et al. 2020) and elevated ferric chelate reductase activity in various Strategy I plants (Kabir et al. 2020). Additionally, AM fungi have been shown to upregulate Fe-related genes such as IRT1 and FRO1 in sunflower and Medicago sativa (Kabir et al. 2020; Rahimi et al. 2021; Rahman et al. 2020), and genes involved in Fe acquisition and transport (NAS, YS1, and OPT8) in sorghum and maize (Kobae et al. 2014; Prity et al. 2020). 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 (Fann et al. 2021, Rai et al. 2021), this study also aimed to analyze how AM symbiosis modulates the impact of Fe deficiency on the tomato ionome. 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 hours. Afterwards seeds were germinated under sterile conditions on wet filter paper in Petri plates in darkness at 25 °C for 4 days. 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 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)/8h dark (20 °C). They were watered twice a week with a modified half-strength Hoagland solution containing 0.125 mM KH 2 PO 4 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 ccc1 Δ and pmr1 Δ mutants of Saccharomyces cerevisiae were used in this work. ccc1 Δ lacks the vacuolar membrane transporter Ccc1 (Li et al. 2001) and pmr1 Δ 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 were oven-dried at 65 °C for two days, ground to a fine powder, ashed at 550 °C and digested in H 2 O/HNO₃. Tissue nutrients concentrations were analysed 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. 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 0.05M Na-cacodylate buffer (pH7.2) 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 sections (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. 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 RT-qPCR are presented in Supplementary Table 1. The specificity of the primer sets was analyzed by PCR amplification of the R. irregularis cDNA. The real-time RT-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 RT-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 -ΔCT 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 (Supplementary 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 (Supplementary 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 ccc1 Δ and pmr1 Δ were transformed with the different pDRf1-VIT constructs or with the empty vector using a lithium acetate-based method (Schiestl & 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 H 2 O and adjusted to a final OD 600 of 1. Then, 5 µL of serial 1:10 dilutions were spotted on the corresponding selective medium. ccc1 Δ transformants were spotted onto SD-URA supplemented or not with 5 mM FeSO 4 . pmr1 Δ transformed mutants were spotted onto SD without uracil supplemented or not with 2 mM MnCl 2 . 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. 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). 3.2. Arbuscules 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). 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 3-fold reduction in RiEF1α expression was detected in mycorrhizal roots of plants grown under Fe-deficient conditions. 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). 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 (Supplementary 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 proteins 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) (Supplementary Figures 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% (Supplementary 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 (Supplementary Figure 1d). As expected for genes encoding a 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). 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 ( ccc1 Δ). In yeast, ccc1 encodes a vacuolar transporter responsible for Fe sequestration. ccc1 Δ 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 ccc1 Δ cells on SD medium supplemented with 5 mM FeSO 4 . 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 vacuolar Fe transporters (Figure 6a). 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 transport Mn was evaluated using pmr1Δ yeast mutant. 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 on 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 is appears to be specialized in Mn transport. 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 compared to AM colonization. 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. 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. AM colonization partially alleviated these effects. 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 (Supplementary Figure 2). In roots, Fe-deficient conditions affected only the molybdenum content in mycorrhizal plants, without causing statistically significant changes in the concentrations of other nutrients (Supplementary Figure 3). 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 (Supplementary Figure 2). In roots, AM colonization consistently increased copper and Fe contents, regardless of Fe availability. Zinc, manganese, and phosphorus levels also tended to be higher in mycorrhizal roots, although these differences were statistically significant only under Fe-sufficient conditions (Supplementary Figure 3). 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. 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; Jin et al. 2013; Ravet et al. 2009, 2012; 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 to 150 (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 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). 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, suggesting that in the R. irregularis -tomato symbiosis, the fungus functions as a sink for Fe. This observation aligns with 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). 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). 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). 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, the up-regulation of SlFRO1 in mycorrhizal roots is consistent with previous observations in other plant species and supports the view that Fe bioavailability increases in the rhizosphere of mycorrhizal plants (Kabir et al. 2020; Rahimi et al. 2021). SlFRO1 encodes a plasma membrane ferric reductase responsible for increasing Fe availability at the epidermal root cells in Strategy I plants (Robinson et al., 1999). As expected, SlIRT1 expression also increased under these conditions, which may explain the higher Fe content observed in mycorrhizal roots. In contrast, under Fe-sufficient conditions, SlFRO1 transcripts were undetectable and SlIRT1 expression was reduced in mycorrhizal roots, a trend consistent with reports in alfalfa roots (Rahman et al. 2020). Similar patterns of SlFRO1 induction under Fe-deficiency but not under under Fe-sufficient conditions have also been reported in quince and sunflower roots (Kabir et al. 2020; Rahimi et al. 2021). These data reveal that the impact of AM on the Fe-deficiency response in tomato is dependent on Fe availability. Under Fe-limiting conditions, AM enhances the Strategy I Fe acquisition pathway by increasing Fe bioavailability, whereas this effect is not observed when Fe is sufficient. This highlights the context-dependent nature of AM-mediated regulation of Fe homeostasis. The down-regulation of SlIRT1 under optimal conditions suggests that direct Fe uptake through the root epidermis is inhibited in mycorrhizal roots. This down-regulation of the direct uptake pathway in a mycorrhizal root has also been observed 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 grown under optimal Fe conditions suggests that its encoded protein may mediate Fe uptake via the mycorrhizal pathway, potentially explaining the increased Fe content in these roots. Although functional characterization is still pending, SlNRAMP1 is a strong candidate for mediating Fe transport from fungal structures to the plant. The observed down-regulation of SlCHLN , which encodes the enzyme involved in nicotianamine biosynthesis, under Fe-deficient conditions and in mycorrhizal roots agrees with the reduced root to shoot Fe translocation in these conditions. Given the essential role of nicotianamine as a metal chelator facilitating long-distance transport of Fe (Higuchi et al. 1996), a decrease in its biosynthesis could limit the formation of nicotianamie-complexes, thereby restricting Fe mobility from roots to aerial tissues. This idea aligns with previous findings in Arabidopsis , where 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. Further investigation into nicotianamine levels and Fe–nicotianamine complex formation in these conditions would be valuable to confirm this hypothesis and clarify the regulatory network linking Fe homeostasis, nicotianamine biosynthesis, and mycorrhizal symbiosis. 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 yeast complementation assays revealed that SlVIT1, SlVTL1 and SlVTL2 are functional homologues of the yeast vacuolar transporter ccc1, which mediates Fe transport from the cytosol into the vacuole, contributing to Fe detoxification under excess conditions (Li et al. 2001). When expressed in Δpmr1 yeast, all four SlVIT/VTL genes partially rescued growth inhibition under high concentrations of MnCl 2 , suggesting that they mediate manganese transport (Lapinskas et al. 1996). These results indicate that SlVIT1, SlVTL1 and SlVTL2 are capable of transporting both Fe and manganese, while SlVIT2 appears to specifically direct Mn to the Golgi apparatus. 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, expression of the Mn transporter SlVIT2 was up-regulated in mycorrhizal roots and an increased Mn content was found in roots of mycorrhizal plants regardless of Fe availability. Increased manganese tissue concentrations in mycorrhizal plants has been reported in several plant species (Lambert et al. 1979; Eivazi and Weir 1989). The biological relevance of this manganese enrichment may lie in the diverse roles this micronutrient plays in plant physiology, including photosynthesis, respiration, scavenging of reactive oxygen species, pathogen defense and hormone signalling (Alejandro et al. 2020). The observed induction of SlVIT2 in mycorrhizal roots and the associated manganese accumulation point to a potentially important role for this micronutrient in AM. However, further research is needed to clarify the functional significance of SlVIT2 in this context. Regarding the ionomic data, our results indicate that AM colonization partially mitigates the alterations induced in the tomato 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. In conclusion, the data presented in this manuscript demonstrates that Fe availavility is a key factor in 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 the fungus acts as a sink of Fe. 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. 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”. References Alejandro, S., Höller, S., Meier, B,. and Peiter E. 2020. Manganese in plants: from acquisition to subcellular allocation. Frontiers in Plant Science 11: 300. https://doi.org/10.3389/fpls.2020.00300 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. https://doi.org/10.1093/jxb/erp346 Azcón-Aguilar, C., and Barea, J.M. 2015. Nutrient cycling in the mycorrhizosphere. 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(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. Figure 2 . Histochemical Perls staining of transversal root sections of non-mycorrhizal (NM, a and b) and mycorrhizal (Myc, c and d) plants, incubated either with HCl (unstained control, a and c) or the Perls reagent (stained, b and d). The scale bars correspond to 100 μm. Figure 3. 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 -ΔCT 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). Figure 4 . 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 S. 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 -ΔCT 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). Figure 5 . 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 -ΔCT method, with SlEF1α and S lACTIN as normalizers. Data represent means ± standard error (n = 3). Different letters indicate statistically significant differences between treatments (p < 0.05, Duncan’s test). Figure 6 . Functional analysis of S. 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 FeSO 4 . (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 MnCl 2 . Figure 7 . Effect of Fe deficiency and mycorrhizal colonization on the S. 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. Right panels: Heat maps and hierarchical clustering of nutrient contents across treatments. Information & Authors Information Version history V1 Version 1 08 September 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Plant, Cell & Environment Keywords arbuscular mycorrhiza gene expression growth ionome iron transcriptome Authors Affiliations Víctor M. López-Lorca Estacion Experimental del Zaidin View all articles by this author Olga López-Castillo Estacion Experimental del Zaidin View all articles by this author Mª Jesús Molina-Luzón Estacion Experimental del Zaidin View all articles by this author Nuria Ferrol 0000-0001-7963-3537 [email protected] Estacion Experimental del Zaidin View all articles by this author Metrics & Citations Metrics Article Usage 282 views 149 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Víctor M. López-Lorca, Olga López-Castillo, Mª Jesús Molina-Luzón, et al. Iron Availability Modulates Arbuscular Mycorrhizal Symbiosis, Which in Turn Influences the Expression of Iron-Responsive Genes and Nutrient Balance in Solanum lycopersicum. Authorea . 08 September 2025. DOI: https://doi.org/10.22541/au.175732766.62550850/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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