Transcriptomic analysis reveals the participation of NTRC in iron homeostasis in Arabidopsis

ORIGINAL RESEARCH Transcriptomic analysis reveals the participation of NTRC in iron homeostasis in Arabidopsis Fernando Rodríguez-Marín 1,2 | Juan M. Pérez-Ruiz 1,2 | Francisco J. Cejudo 1,2 1 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla and CSIC, Sevilla, Spain 2 Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain Correspondence Francisco J. Cejudo, Email: [email protected] Juan M. Pérez-Ruiz, Email: [email protected] Funding information Ministerio de Ciencia e Innovación, y Universidades (MICIU)/Agencia Estatal de Investigación (AEI), Grant/Award Numbers: PID2020-115156GB-I00, PID2023-146573NB-I00 Edited by Y. Allahverdiyeva Abstract NADPH-dependent thioredoxin reductase C (NTRC) plays a central role in redox regulation of chloroplast photosynthetic metabolism. Accordingly, Arabidopsis (Arabidopsis thaliana) NTRC-null mutants show defective photosynthetic performance and growth inhibition. Remarkably, these mutants show almost a wild-type phenotype at the seedling stage, which raises the question of whether NTRC plays different functions throughout plant development. In this work, we have addressed this issue by performing transcriptome comparisons of Arabidopsis wild-type and ntrc mutant lines at seedling and adult stages of development. In contrast with the high impact of NTRC on leaves from adult plants, the low transcriptomic differences in seedlings suggested a less relevant function of NTRC at this stage of plant development. Notably, the ntrc mutant showed transcriptomic changes resembling the response to Fe excess throughout plant development, though this response was almost unique at the seedling stage. The lack of NTRC caused altered levels of Mn, Zn, Cu, S, P, K and Na, but no significant differences in the content of Fe, as compared with the wild type. Moreover, at the seedling stage, the lack of NTRC caused hypersensitivity to Fe deficit but a protective effect in response to Fe excess, most likely due to lower ROS accumulation in the mutant seedlings. Our results reveal the different impacts of NTRC throughout plant development and identify Fe homeostasis as a process highly affected by NTRC, most notably at the seedling stage. 1|INTRODUCTION Plant chloroplasts, the organelles that perform photosynthesis, are the source of metabolic intermediates that support plant growth. Furthermore, these organelles have an important sensor activity able to monitor changes in environmental conditions, which influence photosynthesis performance (Schwenkert et al. 2022; Tano and Woodson 2022). A central regulatory mechanism that allows the rapid response of chloroplast photosynthetic performance to environmental cues, such as light intensity and darkness, is based on the protein dithiol-disulfide exchange, which is the basis of thiol-dependent redox regulation (Mittler and Jones 2024; Sies et al. 2024). This regulatory mechanism, which largely relies on the protein disulfide reductase activity of thioredoxins (TRXs), is universally found in any type of organism, from bacteria and fungi to animals and plants. In contrast with heterotrophic organisms, which harbour two, at most three, TRXs that use reducing power from NADPH via the participation of an NADPH-dependent TRX reductase (NTR), the TRX gene family in plants is remarkably complex (Meyer et al. 2012). Among plant cell compartments, chloroplasts are the organelles with Received: 10 January 2025 Accepted: 18 March 2025 DOI: 10.1111/ppl.70203 Physiologia Plantarum This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2025 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society. Physiologia Plantarum. 2025;177:e70203. wileyonlinelibrary.com/journal/ppl 1of14 https://doi.org/10.1111/ppl.70203 the highest content of TRXs (Zaffagnini et al. 2019; Geigenberger et al. 2017), which are grouped into typical and atypical isoforms (Chibani et al. 2021). Though it remains to be established the source of reducing power for all chloroplast TRXs, it is known that typical TRXs, such as those of the types mand f, which are the most abundant in plant chloroplasts (Okegawa and Motohashi 2015), are reduced by ferredoxin (FDX), the final acceptor of the photosynthetic electron transport chain via the action of a FDX-dependent TRX reductase (FTR) (Schürmann and Buchanan 2008). Thus, the FDXFTR-TRXs redox system links the redox regulation of chloroplast enzymes to light. Chloroplasts harbour an additional redox system, NTRC, an NTR with a TRX domain at the C-terminus (Serrato et al. 2004), which has a high affinity for NADPH (Bernal-Bayard et al. 2012). The finding that the hydrogen peroxide scavenging enzyme 2-Cys peroxiredoxin (PRX) is efficiently reduced by NTRC (Alkhalfioui et al. 2007; Moon et al. 2006; Pérez-Ruiz et al. 2006), led to the proposal of an antioxidant function for this enzyme. However, the Arabidopsis ntrc mutant, which is devoid of NTRC, shows a rather severe growth inhibition phenotype (Lepistö et al. 2009; Pérez-Ruiz et al. 2006; Serrato et al. 2004), and impairment of different redoxregulated processes supporting the proposal that NTRC acts as a central hub in chloroplast redox regulation, as recently reviewed (Cejudo et al. 2021). The finding that Arabidopsis mutants combining the deficiencies of NTRC and 2-Cys PRXs recover a WT-like phenotype uncovered the essential function of 2-Cys PRXs in controlling the reducing capacity of chloroplast TRXs, which allows optimization of the photosynthetic performance of the organelle in response to changes in light intensity (Pérez-Ruiz et al. 2017; Yokochi et al. 2021). Moreover, NTRC is essential for the activity of TRXs of the types xand fduring the early stages of plant development (Ojeda et al. 2017). However, ntrc seedlings develop normal green cotyledons (Lepistö et al. 2009; Ojeda et al. 2021), and neither the lack nor the overexpression of NTRC has any effect on embryogenesis in developing seeds (GallardoMartínez et al. 2023) in contrast with its relevant role at latter stages of plant development. Hence, NTRC has differential functions throughout plant development: a poor contribution at early stages of plant development when chloroplast biogenesis occurs, whereas in fully differentiated chloroplasts from mature leaves, this enzyme plays a key role in the redox regulation of photosynthesis in response to changes in light intensity. Despite the extensive analysis of the function of NTRC in adult leaves, hence, with fully differentiated chloroplasts, the function of this enzyme at earlier stages of plant development remains poorly known. In this work, we have addressed this issue by performing a comparative analysis of the transcriptomes of the Arabidopsis ntrc mutant, as compared to WT, in seedlings and leaves from adult plants. Our results show a low transcriptomic impact of the lack of NTRC at the seedling stage, in contrast to the severe impact on adult leaves. Remarkably, these analyses revealed that the lack of NTRC affects the expression of genes involved in Fe homeostasis, triggering transcriptomic changes resembling the response to Fe excess, which is more relevant at the seedling stage. 2|MATERIALS AND METHODS 2.1 |Biological material, growth conditions, and Fe treatments Arabidopsis thaliana WT (ecotype Columbia) and ntrc mutant (Serrato et al. 2004) were routinely grown in soil in growth chambers under short-day (8/16 h light/ dark) photoperiod at 22 and 20C during light and dark periods, respectively, and light intensity of 125 μmol m 2 s 1 . WT and ntrc seedlings were grown on Murashige and Skoog (MS) medium containing 0.35% (w/v) Gelrite (Duchefa) under continuous light at 22C and 125 μmol m 2 s 1 . Fe treatments were performed in synthetic media (pH 5.5, 0.7% agar) containing 5 mM KNO 3 , 2.5 mM KH 2 PO 4 , 2 mM MgSO 4 ,2mM Ca(NO 3 ) 2 ,70μMH 3 BO 3 ,14μM MnCl 2 ,10μM NaCl, 1 μM ZnSO 4 , 0.5 μM CuSO 4 , 0.2 μMNa 2 MoO 4 , 4.7 mM MES (Rodríguez-Celma et al. 2013) and a variable concentration of Fe-EDTA as source of Fe. For Fe treatments at the seedling stage, seeds were germinated in synthetic media containing either 50 μM Fe-EDTA (Fe-sufficient, control condition), 500 μM Fe-EDTA (Fe-excess), or medium without FeEDTA and supplemented with 100 μM ferrozine (sodium 4-[3-(pyridin2-yl)-6-(4-sulfophenyl)-1,2,4-triazin-5-yl]benzene-1-sulfonate) to chelate residual Fe (Fe-deficient). Fe treatments at the adult stage were performed on plants grown in Fe-sufficient medium for 21 days and transferred to Fe-sufficient (control conditions), Fe-excess or Fe-deficient media for 9 days. 2.2 |RNA extraction, RNA-Seq and RT-qPCR analyses Two different transcriptomics experiments, at the adult and seedling stages of plant development, were carried out. For adult samples, eight-week-old short-day grown WT and ntrc plants were randomly selected, and young rosette leaves were collected during the day period (after 2 h of illumination at 125 μmol m 2 s 1 ). The experimental design consisted of three biological replicates for each genotype, each of them containing leaves from three individual plants. WT raw RNA-Seq data from the adult stage can be found in Gene Expression Omnibus, identified with accession number GSE147793. RNA extraction was performed from pooled leaf samples using the Sure Prep kit (Fisher), following the manufacturer's instructions. RNA concentration and purity were tested by an Agilent 2100 Bioanalyzer, a microfluidics-based platform that performs quality control of DNA and RNA samples before sequencing. Library construction of cDNA molecules was carried out following the manufacturer's instructions using the TruSeq Stranded Total RNA with Ribo-Zero kit (Illumina). The generated DNA fragments were sequenced with the Illumina HiSeq 4000 platform at STAB-VIDA (Caparica, Portugal), yielding approximately 60–80 million 150 base pairs long paired-end reads for each sample. For seedling samples, six-day-old WT and ntrc mutant seedlings, grown under continuous light in MS medium and lacking true leaves, were collected. The experimental design consisted of 2of14 RODRÍGUEZ-MARÍN ET AL. Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License three biological replicates for each genotype. RNA extraction and quality control were carried out following the above indicated procedure for adult samples. The cDNA libraries were constructed according to the manufacturer's instructions using the Illumina Stranded mRNA prep ligation kit (Illumina). Sequencing of the DNA fragments was performed with the Illumina NextSeq 500 platform at Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER, Seville, Spain), yielding approximately 18–26 million 75 base pairs long single-end reads for each sample. For all samples, the FastQC software package was used to control the quality (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Once the good quality of all samples had been determined, read mapping to The Arabidopsis Col-0 TAIR 10 reference genome (https://plants. ensembl.org/) and transcript assembly were performedwiththesoftware tools HISAT2 and StringTie (Pertea et al. 2016) using default parameters. Differential expression analysis was carried out with the Ballgown (Pertea et al. 2016) and LIMMA (Ritchie et al. 2015) R packages. Differentially expressed genes (DEGs) were selected using a fold change of ± log 2 (2) and P-value <0.05 computed according to a moderated t-test. Volcano plots and Venn diagrams were generated using the R packages ggplot2 and eulerr. Gene Ontology (GO) term enrichment analysis over the different gene sets was performed with the enrichGO function of the Bioconductor R package ClusterProfiler (Yu et al. 2012). From the same R package, the compareCluster function was used to compare the different gene ontology terms of adult and seedling samples. Principal Component analysis was carried out using the prcomp function from the R package stats of the R Core Team. For Real-time quantitative PCR (RT-qPCR) analysis, total RNA was extracted using the Trizol reagent (Invitrogen) and cDNA synthesis was performed with 1 μg of total RNA using the Maxima first-strand cDNA synthesis kit (Thermo Scientific) according to manufacturer's instructions. RT-qPCR was performed using an IQ5 real-time PCR detection system (Bio-Rad) with oligonucleotides listed in Supplemental Table S1. Expression levels were normalized using ACTIN2,UBIQUITIN10 and UBIQUITIN-CONJUGATING ENZYME9 as reference genes. 2.3 |Measurements of chlorophyll levels and determination of photosynthetic parameters Chlorophyll levels were measured as previously described (Pérez-Ruiz et al. 2006). Room temperature chlorophyll afluorescence was measured using a pulse-amplitude modulation fluorometer IMAGINGPAM M-Series (Walz). The maximum quantum yield of PSII was assayed after incubation of plants in the dark for 30 min by calculating the ratio of the variable fluorescence, F v , to maximal fluorescence, F m (F v /F m ). Induction-recovery curves were performed using red (635 nm) actinic light at 81 μmol m 2 s 1 for 10 min. Saturating pulses of red light at 10,000 μmol m 2 s 1 intensity and 0.6 s duration were applied every 60 s, and recovery in darkness was recorded for up to 10 min. The parameter Y(II), corresponding to the respective quantum yield of PSII photochemistry, was calculated according to reported equations (Kramer et al. 2004). 2.4 |Determination of anion superoxide content The detection of superoxide anion content was performed with nitroblue tetrazolium (NBT) staining, as previously described (Cordoba et al. 2016), with some modifications. Briefly, at least six seedlings of WT and ntrc mutant grown in Fe-deficient, Fe-sufficient (Fe-control) and Fe-excess media were randomly selected and incubated in staining buffer (10 mM phosphate pH 7.6, 10 mM NaN 3 and 0.1% NBT (Sigma)) for 2.5 h at room temperature in agitation. Then, the NBT solution was removed, and a bleaching solution (ethanol: acetic acid: glycerol; 3:1:1) was added. Once the chlorophyll had been removed, a new bleaching solution was added to the seedlings, which were stored at 4C until images were taken with an Olympus Microscope. For the quantification of the area of cotyledons stained with NBT, colour thresholds were used in the ImageJ software. 2.5 |Ionomics determinations For WT and the ntrc mutant, whole rosette samples of 8-week-old plants grown in soil under short-day conditions and 4-day-old seedlings grown under continuous light in MS medium were lyophilized for determination of ion content by inductively coupled plasma mass spectrometry (ICP-MS) at the Servicio de Análisis, Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNASE, CSIC), Seville, Spain. In brief, 4 mL of HNO 3 (65%) was added to the weighed lyophilized material. The digestion was carried out under pressure in a microwave oven (MARS ONE CEM). The treated samples were diluted with ultrapure water and passed through nylon filters. Ion determination was performed using an AGILENT 7800 ICP-MS. Principal Component analysis was carried out using the prcomp function from the R package stats of the R Core Team. 2.6 |Statistical analysis Statistical analyses were performed using GraphPad Prism version 6.01 (GraphPad Software, Inc.), and the experimental results were analyzed using two-tailed Student's t-tests. 3|RESULTS 3.1 |Comparative transcriptomic analyses reveal a poor impact of NTRC at the seedling stage of plant development With the aim of determining the impact of NTRC at different stages of plant development, we have performed a comparative RODRÍGUEZ-MARÍN ET AL.3of14 Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License transcriptomic analysis of Arabidopsis WT and the ntrc mutant at seedling and adult stages. To that end, genome-wide transcriptomes were determined by RNA-Seq analysis of three independent biological samples harvested from young leaves of 8-week-old Arabidopsis WT and ntrc mutant plants grown under short-day photoperiod (adult stage) and from seedlings that had been grown for 6 days on MS medium under continuous light (seedling stage) (Figure 1A, C). Though the ntrc mutant contained lower chlorophyll levels at both developmental stages, the difference was more significant at the adult stage (Figure 1B, D). The sequencing quality of the samples was assessed by the high percentage of mapped reads and correlation between biological replicates (around 99%) (Figure S1A-D). A principal component analysis showed that the ntrc and WT seedling replicates clustered together, whereas replicates from adult ntrc clustered separately from the WT (Figure S2), thus indicating a more relevant impact of NTRC on the transcriptomes at adult than at seedling stages. The comparison of the transcriptomes from leaves of the adult plants of the WT and the ntrc mutant, using a fold change of gene expression of ±log 2 (2) and p< 0.05, identified a large set of 535 DEGs in the ntrc mutant, 358 upregulated and 177 downregulated (Figure 2A). Strikingly, a relatively low number of DEGs (94), of which 35 were upregulated and 59 downregulated (Figure 2B), were identified in the comparison of the WT and the ntrc mutant transcriptomes at the seedling stage. The lists of upregulated and downregulated genes in the ntrc mutant at both developmental stages are available as supplemental information (Supplemental Appendix S1). A Venn diagram representation of DEGs at adult and seedling stages identified an overlapping set of 24 DEGs common to both stages (Figure 2C). Thus, the higher number of DEGs between the ntrc mutant and the WT at the adult stage confirms the deep impact of NTRC on plant growth, in line with the severe growth inhibition phenotype of the ntrc mutant (Figure 1A, B), whereas the modest transcriptomic changes shown by the ntrc seedlings reveals the poor impact of NTRC at this early developmental stage, which is in line with the almost WT-like phenotype of the ntrc seedlings (Figure 1C, D). Moreover, the low number of DEGs commonly detected at seedling and adult stages indicates that the participation of NTRC throughout plant development is restricted to a few biological processes. 3.2 |The ntrc mutant shows transcriptomic changes resembling the response to Fe excess To identify the processes affected by the lack of NTRC at adult and seedling stages, a gene ontology (GO) term enrichment analysis was performed (Supplemental Appendix S2). In line with the severe growth inhibition phenotype of the ntrc mutant, the comparison of the WT and ntrc mutant transcriptomes at the adult stage showed the pleiotropic effects caused by the absence of NTRC, affecting a wide range of biological processes, as previously reported (Lepistö et al. 2009). Upregulated genes in the ntrc mutant at this developmental stage are enriched in GO terms involved in metabolism (carbohydrates, amino FIGURE 1 Growth phenotypes of Arabidopsis WT and ntrc mutant at adult and seedling stages of development. Representative photographs of WT and ntrc mutant plants grown under short-day photoperiod for 8 weeks (A) and seedlings grown in MS under continuous light for 6 days (C). Chlorophyll levels in WT and ntrc adult leaves (B) and seedlings (D), determined from at least 10 replicates, represented as average values ± SD. Asterisks represent significant differences compared with the WT (*, p< 0.05, ***p< 0.001; Student's t-test). 4of14 RODRÍGUEZ-MARÍN ET AL. Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License acids and tetrapyrroles), abiotic stress response (hypoxia, nutrient level, nitrate, light, heat, metal ion), biotic stress response (insect, salicylic acid), and detoxification (Figure 3A). In contrast, GO terms related to glucosinolate and glycerolipid metabolisms, cell wall organization, cellular homeostasis, response to starvation and ion transport were the biological processes identified among the downregulated genes in the ntrc mutant (Figure 3B). At the seedling stage, most enriched GO terms among the upregulated genes in the ntrc mutant were related to metal ion homeostasis (Figure 3C), whereas GO terms associated with cellular homeostasis, response to starvation and ion transport, as well as response to metal ion and jasmonic acid biosynthesis, were identified among the downregulated genes (Figure 3D). To further analyze the impact of NTRC at different stages of plant development, Venn diagrams were constructed identifying common GO terms among upregulated and downregulated genes in the ntrc mutant at adult and seedling stages (Figure 4A, B and Supplemental Appendix S3). This analysis identified GO terms related to detoxification and homeostasis of Fe enriched at both stages of development (Figure 4C). The fact that many of the DEGs between the ntrc mutant and the WT from adult and seedling stages identified terms such as “Intracellular iron homeostasis”,“Iron ion homeostasis”,“Multicellular organismal-level iron homeostasis”,“Response to iron ion”and “Response to iron starvation”(Figure 4C) revealed the effect of NTRC on the transcription of genes involved in Fe homeostasis at both developmental stages here analyzed, a previously unrecognized function of NTRC, which seems more relevant in seedlings than in leaves from adult plants. In plants, Fe homeostasis involves different organs and tissues including roots and the vascular system and, in leaf photosynthetic cells, different cell compartments (Liang, 2022). Thus, to figure out the effect of NTRC on Fe homeostasis at different developmental stages, DEGs involved in Fe homeostasis or response to Fe in the transcriptomic analyses of seedling and adult samples were selected, and their fold-changes in the level of expression were represented in different organs and cell compartments (Figure 5A). Genes encoding peptides (FEP1, FEP2, IMA4, IMA6) that positively regulate the transcription factors bHLH38, bHLH39, bHLH100 and bHLH101, all of them involved in activation of the expression of genes responsive to Fe deficiency, were found among the most downregulated genes in seedlings and, at lower extent, in adult leaves of the ntrc mutant (Figure 5A), hence indicating that the lack of NTRC triggers transcriptomic changes resembling the response to Fe excess. This notion was further supported by the identification of genes involved in the transport of Fe through the phloem and xylem, such as OLIGO PEPTIDE TRANSPORTER 3 (OPT3) among the downregulated genes at both stages. Furthermore, genes encoding the Fe transporter in root cells IRON REGULATED TRANSPORTER 1 (IRT1) and enzymes of the sideretin biosynthetic pathway were also downregulated in seedlings of the ntrc mutant (Figure 5A). These genes showed differences only at the seedling stage due to the nature of the adult samples, which excluded root tissues. In leaves cells, the comparative transcriptomic analyses revealed upregulation of genes encoding transport of Fe to the vacuoles, VACUOLAR IRON TRANSPORTER-LIKE 1,2and 5(VTL1, FIGURE 2 Comparative transcriptomic analysis between the WT and the ntrc mutant at seedling and adult stages of development. RNASeq analyses were carried out with RNA samples extracted from young leaves of 8-week-old Arabidopsis WT and ntrc mutant plants that had been grown under short-day photoperiod (125 μEm 2 s 1 )(adultstage). For the seedling stage, the WT and the ntrc mutant were grown for 6 days on MS medium under continuous light (125 μEm 2 s 1 ). Volcano plots representing differentially upregulated (red), downregulated (blue) and unaltered (black) genes of ntrc versus WT comparisons at adult (A) and seedling (B) stages. (C) Venn diagram indicating the common DEGs between adult and seedlings stages in the ntrc mutant. RODRÍGUEZ-MARÍN ET AL.5of14 Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License VTL2, VTL5) and the chloroplast PERMEASE IN CHLOROPLASTS 1 (PIC1), both at adult and seedling stages (Figure 5A). Furthermore, genes involved in Fe-S assembly (NEET) and tetrapyrrole biosynthesis (HEMA1) into the chloroplast and, remarkably, genes encoding ferritins (FER1,FER3 and FER4) were upregulated in adult and seedlings samples of the ntrc mutant, further supporting the notion that the lack of NTRC triggers a Fe excess transcriptomic response. On the contrary, CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), which is required for growth under Fe starvation (Urzica et al. 2012), was downregulated in the ntrc mutant at both developmental stages. Among genes encoding enzymes involved in the scavenging of reactive oxygen species (ROS), formed in the presence of Fe via the Fenton reaction, stromal APX (sAPX) was upregulated in the ntrc mutant, whereas FeSOD1-3 genes were downregulated in mutant seedlings and FeSOD1 upregulated in adult leaves. Finally, several of the DEGs between the ntrc mutant and the WT were chosen to validate the results of the RNA-Seq data by RT-qPCR analysis. The lower level of transcripts of the bHLH38,bHLH39,FEP1 and OPT3 genes and the higher levels of the FER1 and NEET genes in adult leaves (Figure 5B) and seedlings (Figure 5C) of the ntrc mutant validated the results of the transcriptomic analysis. 3.3 |Effect of Fe excess and deficit treatments on photosynthetic performance To gain a deeper insight into the function of NTRC in metal ion homeostasis, we sought to analyze the effect of Fe deficiency and excess treatments on the ntrc mutant performance at the two developmental stages analyzed. For seedling treatments, WT and ntrc seeds FIGURE 3 Gene Ontology term enrichment analysis of DEGs in the ntrc mutant at seedling and adult stages of plant development. Enriched map plot showing the 50 most enriched gene ontology terms of different sets of genes in ntrc mutant versus WT comparisons at adult (A, B) and seedling (C, D) stages. Biological processes enriched in the ntrc mutant among the upregulated genes at adult (A) and seedling (C) stages and among downregulated genes at adult (B) and seedling (D) stages. Node size represents the number of genes associated with each gene ontology enriched term, with edges connecting overlapping gene sets. Colour gradient from blue to red represents low to high significance levels. GO terms clusters were manually annotated. 6of14 RODRÍGUEZ-MARÍN ET AL. Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License were germinated and grown for 4 days under continuous light in a synthetic medium supplemented with 50 mM Fe-EDTA (Fe-control, sufficiency conditions), 100 mM ferrozine and no added Fe-EDTA (Fe-deficiency conditions) and 500 mM Fe-EDTA (Fe-excess conditions) (Figure 6A). To analyze the effect at adult stage, WT and ntrc plants grown for 21 days under short-day photoperiod and Fesufficient conditions (Fe-control) were transferred to Fe-control, Fedeficiency or Fe-excess synthetic media and grown for 9 days. The photosynthetic parameters Fv/Fm, which reflects PSII stability, and quantum yield of PSII photochemistry (Y(II)) were determined as readouts of the treatments (Figure 6A-C). The Fe deficiency treatment caused no significant differences in the levels of Fv/Fm either at adult or seedling stages of the WT and ntrc mutant (Figure 6A, B). However, the quantum yield of PSII (Y(II)) revealed a slight effect of this treatment at the adult stage in the ntrc mutant, which was more severe at the seedling stage (Figure 6A, C), hence indicating the sensitivity of the ntrc mutant to Fe deficiency, which is higher at early stages of development. The Fe excess treatment, which did not significantly affect the WT at the adult stage, caused slightly higher levels of Fv/Fm (Figure 6A, B), but no significant variation of Y(II) (Figure 6A, C) in adult ntrc plants. In contrast, this treatment had a severe effect on WT seedlings, as revealed by the lower levels of Fv/Fm (Figure 6A, B) and Y(II) (Figure 6A, C). Remarkably, the lack of NTRC triggered a significant tolerance to Fe excess at the seedling stage, as shown by the higher levels of Fv/Fm (Figure 6A, B) and Y(II) (Figure 6A, C) in seedlings of the ntrc mutant. It is known that Fe excess may be potentially toxic for plants by triggering ROS formation by the Fenton reaction (Halliwell and Gutteridge 1992). Thus, the better performance of the ntrc mutant seedlings under Fe excess might be due to altered ROS homeostasis caused by the lack of NTRC. To test this possibility, seedlings of the WT and the ntrc mutant subjected to Fe excess and deficiency FIGURE 4 GO term enrichment analysis of common DEGs in seedling and adult samples of the ntrc mutant. Venn diagram of GO terms enriched among upregulated (A) and downregulated (B) genes in the ntrc mutant at adult and seedling stages as compared with the WT. (C) Dot plot representing common GO terms at adult and seedling stages. Dot size represents the ratio of genes associated with the GO term among the total number of DEGs. Colour gradient from blue to red represents low to high significance levels. RODRÍGUEZ-MARÍN ET AL.7of14 Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License treatments were stained with NBT to detect the contents of superoxide anion (Figure 7A). No significant differences in NBT staining between the WT and ntrc mutant seedlings were observed under control conditions or in response to Fe deficiency; in contrast, the treatment with Fe excess caused higher levels of NBT staining, hence of superoxide anion, in WT, but not in the ntrc mutant seedlings FIGURE 5 Summary of the fold-changes in the expression of selected DEGs involved in Fe homeostasis in adult and seedling stages and validation of the RNA-Seq data. (A) A colour gradient from blue to red through white is used to represent changes of expression, represented as log 2 (fold-change), of genes encoding proteins related to Fe homeostasis selected from the comparative transcriptomic analyses between the WT and ntrc mutant at seedling (squares) and adult (circles) stages of development. Proteins are placed in their predicted cell compartments (chloroplast, vacuole and nucleus) and tissues (roots and vascular). (B, C) RT-qPCR analysis of selected genes upregulated or downregulated in the ntrc mutant. Transcript levels of selected genes specifically upregulated (FERRITIN1 and NEET) or downregulated (bHLH38, bHLH39, FEP1 and OPT3)inthentrc mutant, according to RNA-Seq data, were determined by RT-qPCR using RNA isolated from young leaves of plants grown under short-day conditions for 8 weeks (B) or from seedlings grown under continuous light for 6 days (C). For each gene, the levels of transcripts were normalized against three reference genes (as indicated in Materials and methods) and referred to their levels of expression in the WT, which was arbitrarily considered as 100%. Values represent the mean ± SD of three technical replicates. Gene-specific oligonucleotides are listed in Table S1. 8of14 RODRÍGUEZ-MARÍN ET AL. Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 6 Legend on next page. RODRÍGUEZ-MARÍN ET AL.9of14 Physiologia Plantarum 13993054, 2025, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/ppl.70203 by Readcube (Labtiva Inc.), Wiley Online Library on [21/04/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License