Iron is an essential micronutrient for all eukaryotic organisms because it participates as a redox cofactor in multiple metabolic processes. Iron bioavailability is highly restricted due to the low solubility of its oxidized form, frequently leading to iron deficiency anemia. The baker’s yeast Saccharomyces cerevisiae is used as a model organism for iron homeostasis studies, but also as a food supplement and fermentative microorganism in the food industry. Yeast cells use the vacuolar Ccc1 transporter to detoxify and store excess iron in the vacuoles. Here, we modulate CCC1 expression and properties to increase iron extraction from the environment. We show that constitutive expression of full-length CCC1 is toxic, whereas deletion of its cytosolic amino-terminal (Nt) domain (NtΔCCC1) rescues this phenotype. Toxicity is exacerbated in cells lacking AFT1 transcription factor. Further characterization of NtΔCcc1 protein suggests that it is a partially functional protein. Western blot analyses indicate that deletion of Ccc1 Nt domain does not significantly alter GFP-Ccc1 protein stability. A functional full-length GFP-Ccc1 protein localized to particular regions of the vacuolar membrane, whereas GFP-NtΔCcc1 protein was evenly distributed throughout this endogenous membrane. Interestingly, expression of NtΔCCC1 increased the accumulation of endogenous iron in cells cultivated under iron-sufficient conditions, a strategy that could be used to extract iron from media that are not rich in iron., This research was funded by a predoctoral contract ACIF/2018/077 (to R.S.-D.) and grant PROMETEO/2020/014 from the Regional Government of Valencia (Generalitat Valenciana), grant BIO2017-87828-C2-1-P and PID2020-116940RB-I00 from the Spanish Ministry of Science, Innovation and Universities, and FEDER funds (ESF, European Social Fund)., Peer reviewed

Iron is a vital micronutrient that functions as an essential cofactor in multiple biological processes, including oxygen transport, cellular respiration, and metabolic pathways, such as sterol biosynthesis. However, its low bioavailability at physiological pH frequently leads to nutritional iron deficiency. The yeast Saccharomyces cerevisiae is extensively used to study iron and lipid metabolisms, as well as in multiple biotechnological applications. Despite iron being indispensable for yeast ergosterol biosynthesis and growth, little is known about their interconnections. Here, we used lipid composition analyses to determine that changes in the pattern of sterols impair the response to iron deprivation of yeast cells. Yeast mutants defective in ergosterol biosynthesis display defects in the transcriptional activation of the iron-acquisition machinery and growth defects in iron-depleted conditions. The transcriptional activation function of the iron-sensing Aft1 factor is interrupted due to its mislocalization to the vacuole. These data uncover novel links between iron and sterol metabolisms that need to be considered when producing yeast-derived foods or when treating fungal infections with drugs that target the ergosterol biosynthesis pathway., This research was supported by grants BIO2017-87828-C2-1-P, PID2020-116940RB-I00, and RED2018-102467-T funded by MCIN/AEI/10.13039/501100011033 and, in the case of BIO2017-87828-C2-1-P, by ERDF A way of making Europe, and ACIF/2019/214 predoctoral fellowship funded by “Generalitat Valenciana”., Peer reviewed

Copper and iron proteins play a wide range of functions in living organisms. Metal assembly into metalloproteins is a complex process, where mismetalation is detrimental and energy-consuming to cells. Under metal deficiency, metal distribution is expected to reach a metalation ranking, prioritizing essential versus dispensable metalloproteins, while avoiding interferences with other metals and protecting metal-sensitive processes. In this review, we propose that posttranscriptional Modulators of Metalloprotein messenger RNA (ModMeR) are good candidates in metal prioritization under metal-limited conditions. ModMeR target high quota or redundant metalloproteins and, by adjusting their synthesis, ModMeR act as internal metal distribution valves. Unappropriate metalation of ModMeR targets could compete with metal delivery to essential metalloproteins and interfere with metal-sensitive processes, such as chloroplastic photosynthesis and mitochondrial respiration. Regulation of ModMeR targets could increase or decrease the metal flow through interconnected pathways in cellular metal distribution, helping to adequate differential metal requirements. Here, we describe and compare ModMeR that function in response to copper and iron deficiencies. Specifically, we describe copper-microRNAs from Arabidopsis thaliana and diverse iron ModMeR from yeast, mammals and bacteria, under copper and iron deficiencies, as well as the influence of oxidative stress. Putative functions derived from their role as ModMeR are also discussed., This work was supported by grants BIO2017-87828-C2-1-P and PID2020-116940RB-I00 funded by MCIN/AEI/10.13039/501100011033 and, in the case of BIO2017-87828-C2-1-P, by ERDF A way of making Europe., Peer reviewed

Eukaryotic cells rely on iron as an indispensable cofactor for multiple biological functions including mitochondrial respiration and protein synthesis. The budding yeast Saccharomyces cerevisiae utilizes both transcriptional and posttranscriptional mechanisms to couple mRNA levels to the requirements of iron deprivation. Thus, in response to iron deficiency, transcription factors Aft1 and Aft2 activate the expression of genes implicated in iron acquisition and mobilization, whereas two mRNA-binding proteins, Cth1 and Cth2, posttranscriptionally control iron metabolism. By using a genome-wide approach, we describe here a global stabilization of mRNAs, including transcripts encoding ribosomal proteins (RPs), when iron bioavailability diminishes. mRNA decay assays indicate that the mRNA-binding protein Pub1 contributes to RP transcript stabilization during adaptation to iron limitation. In fact, Pub1 becomes critical for growth and translational repression in low-iron conditions. Remarkably, we observe that pub1Δ cells also exhibit an increase in the transcription of RP genes that evidences the crosstalk between transcription and degradation mechanisms to maintain the appropriate mRNA balance under iron deficiency conditions., This work was supported by grants PID2020-116940RB-I00 to S. P. and RED2018-102467-T and PID2020-112853GB-C31 to J.E.P.-O., all of them funded by MCIN/AEI/10.13039/501100011033., Peer reviewed

Ergosterol is a specific sterol component of yeast and fungal membranes. Its biosynthesis is one of the most effective targets for antifungal treatments. However, the emergent resistance to multiple sterol-based antifungal drugs emphasizes the need for new therapeutic approaches. The allylamine terbinafine, which selectively inhibits squalene epoxidase Erg1 within the ergosterol biosynthetic pathway, is mainly used to treat dermatomycoses, whereas its effectiveness in other fungal infections is limited. Given that ergosterol biosynthesis depends on iron as an essential cofactor, in this report, we used the yeast Saccharomyces cerevisiae to investigate how iron bioavailability influences Erg1 expression and terbinafine susceptibility. We observed that both chemical and genetic depletion of iron decrease ERG1 expression, leading to an increase in terbinafine susceptibility. Deletion of either ROX1 transcriptional repressor or CTH1 and CTH2 post-transcriptional repressors of ERG1 expression led to an increase in Erg1 protein levels and terbinafine resistance. On the contrary, overexpression of CTH2 led to the opposite effect, lowering Erg1 levels and increasing terbinafine susceptibility. Although strain-specific particularities exist, opportunistic pathogenic strains of S. cerevisiae displayed a response similar to the laboratory strain. These data indicate that iron bioavailability and particular regulatory factors could be used to modulate susceptibility to terbinafine., This research was supported by grant PID2020-116940RB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and predoctoral fellowship ACIF/2019/214 funded by ‘Generalitat Valenciana’. We also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Peer reviewed

Iron is an indispensable element that participates as an essential cofactor in multiple biological processes. However, when present in excess, iron can engage in redox reactions that generate reactive oxygen species that damage cells at multiple levels. In this report, we characterized the response of budding yeast species from the Saccharomyces genus to elevated environmental iron concentrations. We have observed that S. cerevisiae strains are more resistant to high-iron concentrations than Saccharomyces non-cerevisiae species. Liquid growth assays showed that species evolutionarily closer to S. cerevisiae, such as S. paradoxus, S. jurei, S. mikatae, and S. arboricola, were more resistant to high-iron levels than the more distant species S. eubayanus and S. uvarum. Remarkably, S. kudriavzevii strains were especially iron sensitive. Growth assays in solid media suggested that S. cerevisiae and S. paradoxus were more resistant to the oxidative stress caused by elevated iron concentrations. When comparing iron accumulation and sensitivity, different patterns were observed. As previously described for S. cerevisiae, S. uvarum and particular strains of S. kudriavzevii and S. paradoxus became more sensitive to iron while accumulating more intracellular iron levels. However, no remarkable changes in intracellular iron accumulation were observed for the remainder of species. These results indicate that different mechanisms of response to elevated iron concentrations exist in the different species of the genus Saccharomyces., This research was supported by grants BIO2017-87828-C2-1-P, PID2020-116940RB-I00, and RED2018-102467-T funded by MCIN/AEI/10.13039/501100011033 and, in the case of BIO2017-87828-C2-1-P, by ERDF A way of making Europe, and by PROMETEO/2020/014 grant from the Regional Government of Valencia (Generalitat Valenciana). Some computations were performed on Tirant III of the Spanish Supercomputing Network (“Servei d’Informàtica de la Universitat de València”) under the project BCV-2021-1-0001 granted to D.P., while others were performed on resources provided by UNINETT Sigma2—the National Infrastructure for High Performance Computing and Data Storage in Norway, project NN8029K. This work has also been supported by a predoctoral fellowship ACIF/2018/077 to R.S-D. and a predoctoral fellowship ACIF/2019/214 to T.J., both funded by Generalitat Valenciana and European Social Fund (ESF). D.P. is a researcher funded by the Research Council of Norway (RCN) grant Nos. RCN 324253 and Distinguished Researcher funded by Generalitat Valenciana plan GenT grant No. CIDEGENT/2021/039., Peer reviewed

Iron participates as an essential cofactor in the biosynthesis of critical cellular components, including DNA, proteins and lipids. The ergosterol biosynthetic pathway, which is an important target of antifungal treatments, depends on iron in four enzymatic steps. Our results in the model yeast Saccharomyces cerevisiae show that the expression of ergosterol biosynthesis (ERG) genes is tightly modulated by iron availability probably through the iron-dependent variation of sterol and heme levels. Whereas the transcription factors Upc2 and Ecm22 are responsible for the activation of ERG genes upon iron deficiency, the heme-dependent factor Hap1 triggers their Tup1-mediated transcriptional repression. The combined regulation by both activating and repressing regulatory factors allows for the fine-tuning of ERG transcript levels along the progress of iron deficiency, avoiding the accumulation of toxic sterol intermediates and enabling efficient adaptation to rapidly changing conditions. The lack of these regulatory factors leads to changes in the yeast sterol profile upon iron-deficient conditions. Both environmental iron availability and specific regulatory factors should be considered in ergosterol antifungal treatments., This research was supported by grant PID2020-116940RB-I00 funded by MCIN/AEI/10.13039/501100011033 to Sergi Puig, grants PID2020-120066RB-I00 funded by MCIN/AEI/10.13039/501100011033 and AICO/2020/086 by ‘Generalitat Valenciana’ to Paula Alepuz, and grant RED2018-102467-T funded by MCIN/AEI/10.13039/501100011033 to Sergi Puig and Paula Alepuz. Tania Jordá was a recipient of a predoctoral fellowship ACIF/2019/214 funded by ‘Generalitat Valenciana’, and Marina Barba-Aliaga was a recipient of a predoctoral fellowship (FPU2017/03542) funded by MCIN/AEI/10.13039/501100011033 and by ESF Investing in your future. The authors also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)., Peer reviewed

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency., This work was supported by grants PID2020-116940RB-I00 and CEX2021-001189-S funded by MCIN/AEI/10.13039/501100011033. T. J. received the ACIF/2019/214 predoctoral fellowship from “Generalitat Valenciana”. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research., Peer reviewed

Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast Saccharomyces cerevisiae have shown that bulk translation is inhibited during adaptation to iron deficiency, which is consistent with its requirement for ribosome biogenesis and recycling. Although iron deficiency anemia is the most common human nutritional disorder, how iron modulates translation in mammals is poorly understood. Studies during erythropoiesis have shown that iron bioavailability is coordinated with globin synthesis via bulk translation regulation. However, little is known about the control of translation during iron limitation in other tissues. Here, we investigated how iron depletion affects protein synthesis in human osteosarcoma U-2 OS cells. By adding an extracellular iron chelator, we observed that iron deficiency limits cell proliferation, induces autophagy, and decreases the global rate of protein synthesis. Analysis of specific molecular markers indicates that the inhibition of bulk translation upon iron limitation occurs through the eukaryotic initiation factor eIF2α and mechanistic target of rapamycin (mTOR) pathways. In contrast to other environmental and nutritional stresses, iron depletion does not trigger the assembly of messenger ribonucleoprotein stress granules, which typically form upon polysome disassembly., This research was supported by grant PID2020-116940RB-I00 and CEX2021-001189-S funded by MCIN/AEI/https://doi.org/10.13039/501100011033, and PRX21/00100 Fulbright Mobility Program Fellowship from Spanish Ministerio de Universidades to S.P., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S), Peer reviewed

Iron serves as a cofactor for enzymes involved in several steps of protein translation, but the control of translation during iron limitation is not understood at the molecular level. Here, we report a genome-wide analysis of protein translation in response to iron deficiency in yeast using ribosome profiling. We show that iron depletion affects global protein synthesis and leads to translational repression of multiple genes involved in iron-related processes. Furthermore, we demonstrate that the RNA-binding proteins Cth1 and Cth2 play a central role in this translational regulation by repressing the activity of the iron-dependent Rli1 ribosome recycling factor and inhibiting mitochondrial translation and heme biosynthesis. Additionally, we found that iron deficiency represses MRS3 mRNA translation through increased expression of antisense long non-coding RNA. Together, our results reveal complex gene expression and protein synthesis remodeling in response to low iron, demonstrating how this important metal affects protein translation at multiple levels., This work was supported by NIH grants AG058713 and AG066704 to V.M.L., and grants PID2020-116940RB-I00 and CEX2021-001189-S from MCIN/AEI/10.13039/501100011033 to S.P. V.N.G. was supported by NIA grants., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX 2021-001189-S), Peer reviewed

Living organisms have developed finely regulated homeostatic networks to mitigate the effects of environmental fluctuations in transition metal micronutrients, including iron, zinc, and copper. In Saccharomyces cerevisiae, the tandem zinc-finger protein Cth2 post-transcriptionally regulates gene expression under conditions of iron deficiency by controlling the levels of mRNAs that code for non-essential ferroproteins. The molecular mechanism involves Cth2 binding to AU-rich elements present in the 3' untranslated region of target mRNAs, negatively affecting their stability and translation. Arabidopsis thaliana has two TZF proteins homologous to yeast Cth2, C3H14 and C3H15, which participate in cell wall remodelling. The present work examines the expression of representative metal homeostasis genes with putative AREs in plants with altered levels of C3H14 and C3H15 grown under varying metal availabilities. The results suggest that C3H15 may act as a post-transcriptional plant modulator of metal adequacy, as evidenced by the expression of SPL7, the main transcriptional regulator under copper deficiency, and PETE2, which encodes plastocyanin. In contrast to S. cerevisiae, the plant C3H15 affects copper and zinc homeostasis rather than iron. When grown under copper-deficient conditions, adult C3H15OE plants exhibit lower chlorophyll content and photosynthetic efficiency compared to control plants, suggesting accelerated senescence. Likewise, metal content in C3H15OE plants under copper deficiency shows altered mobilization of copper and zinc to seeds. These data suggest that the C3H15 protein plays a role in modulating both cell wall remodelling and metal homeostasis. The interaction between these processes may be the cause of altered metal translocation., This research was funded by the Spanish Ministry of Economy and Competitiveness "PID2020-116940RB-I00 and PID2023-148124OB-I00 funded by MCIN/AEI/10.13039/501100011033" and CIGE/2021/092 funded by Generalitat Valenciana., Peer reviewed

Iron is an essential micronutrient for eukaryotic organisms. In response to iron deficiency, the yeast Saccharomyces cerevisiae optimizes iron utilization by downregulating nonessential iron-dependent processes, such as mitochondrial respiration. This regulatory mechanism is mediated by a mRNA-binding protein designated Cth2. In response to iron scarcity, Cth2 binds through its tandem zinc-finger (TFZ) domain to multiple mRNAs encoding proteins that are necessary for iron-dependent pathways. This binding limits the expression of these mRNAs by promoting their degradation and inhibiting their translation. In this study, we have examined a set of wild yeast strains that share a G195R mutation within the Cth2 TZF domain. By genetically editing both laboratory and wild yeast strains, we demonstrate that the Cth2-G195R protein is defective in binding and degradation of its target transcripts, and it accumulates in the nucleus of the cell, leading to a significant growth defect in iron-deficient conditions. Some of these wild yeast strains also display enhanced tolerance to high iron conditions, indicating that they have adapted to environments with elevated iron levels and have consequently diminished their capacity to grow in iron-limiting conditions. These findings highlight the crucial function of Cth2 in enabling yeast cells to adapt to iron-deficient environments., This research was supported by the MCIN/AEI/10.13039/501100011033 under grants PID2020-116940RB-I00, PID2023-148124OB-I00 and CEX2021-001189-S, and by the Spanish Ministry of Science and Innovation under the predoctoral fellowship PRE2021-096933., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S), Peer reviewed

All data relating to this manuscript can be found within the manuscript and its supplementary files. Data that support the findings of this study are available from the corresponding authors upon reasonable request. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifier PXD057108., Copper (Cu) deficiency compromises plant growth and limits crop productivity. Plants respond to Cu scarcity by activating the expression of several miRNAs, known as Cu-miRNAs, which degrade mRNAs from various cuproproteins to conserve Cu. Cu-miRNAs, like most plant miRNAs, associate with ARGONAUTE 1 (AGO1), the primary effector protein of miRNA-mediated gene silencing pathways, whose function is typically modulated by interacting proteins acting as cofactors. However, how AGO1 is regulated and functions under Cu deficiency remains unknown. Here, we searched for AGO1 interactors in Arabidopsis thaliana plants expressing a functional AGO1 protein tagged with the Twin-Strep-tag (TST) polypeptide, grown under Cu-sufficient or Cu-deficient conditions. TST-AGO1 complexes were affinity purified, and proteins were identified using tandem MS. Interestingly, the iron superoxide dismutase 1 (FeSOD1) encoded by FSD1 was enriched in TST-AGO1 complexes purified from plants grown under Cu deficiency. Moreover, fsd1-2 mutant plants showed reduced levels of AGO1 compared with wild-type plants under Cu sufficiency, while both Cu-miRNA-specific and general AGO1 target mRNAs accumulated to higher levels in fsd1-2 plants under both Cu-deficient and Cu-sufficient conditions compared with wild-type plants. These findings suggest that FeSOD1 is essential for proper AGO1 function, and that its superoxide dismutase activity, which mitigates oxidative stress, enhances AGO1 stability, particularly under Cu deficiency., This work was supported by grants or fellowships from MCIN/AEI/10.13039/501100011033 (co-financed by the European Regional Development Fund) [RTI2018-095118-A-I00, PID2021-122186OB-I00 and RYC-2017-21648 to AC; PID2021-127671NB-I00 to GR; PID2020-116940RB-I00 to LP and APG; BIO2017-83184-R and PID2020-114691RB-I00 to JAD; PRE2019-088439 to AEC], and from the Generalitat Valenciana (CIPROM/2022/21 to GR and JAD)., Peer reviewed

Iron is an essential cofactor involved in cellular processes, including energy generation and the biosynthesis of DNA, proteins, and lipids. The limited solubility of iron at physiological pH frequently results in iron deficiency, thus necessitating sophisticated regulatory mechanisms to maintain iron homeostasis. In Saccharomyces cerevisiae, the transcription factor Aft1 mediates the early response to iron limitation by accumulating in the nucleus and activating the iron regulon, a set of genes involved in iron uptake, utilization and sparing. One of Aft1 targets, CTH2, encodes for a protein that promotes iron economy by post-transcriptionally downregulating non-essential iron-dependent pathways. Yeast cells that exhibit defects in unsaturated fatty acid (UFA) biosynthesis, such as mga2Δ mutants, mislocalize Aft1 to the vacuole under iron-deficient conditions, which impairs activation of the iron regulon. In this study, we show that Cth2, but not other nucleo-cytoplasmic shuttling proteins, also accumulates in the vacuole under simultaneous UFA and iron deficiencies. The deletion of autophagy- and piecemeal microautophagy of the nucleus (PMN)-related genes, including ATG1 and NVJ1, prevents Aft1 vacuolar mislocalization. Furthermore, the subcellular distribution of Nvj1 supports PMN activation under these conditions. Despite preventing vacuolar accumulation, these mutations do not restore the regulatory functions of Aft1 and Cth2, nor do they rescue growth in low-iron conditions. These findings suggest that PMN selectively targets non-functional iron-regulated proteins for degradation when both iron and UFA levels are limiting, serving as a quality control mechanism rather than a pathway for functional recovery. These findings underscore a regulatory layer coordinating nutrient sensing and protein turnover., This research was supported by the MCIN/AEI/10.13039/501100011033 under grants PID2020-116940RB-I00 and PID2023-148124OB-I00 and CEX2021-001189-S, and ACIF/2019/214 predoctoral fellowship funded by “Generalitat Valenciana”., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2021-001189-S)., Peer reviewed

[EN] Protein synthesis is a highly energy-consuming process that is downregulated in response to many environmental stresses or adverse conditions. Studies in the yeast Saccharomyces cerevisiae have shown that bulk translation is inhibited during adaptation to iron deficiency, which is consistent with its requirement for ribosome biogenesis and recycling. Although iron deficiency anemia is the most common human nutritional disorder, how iron modulates translation in mammals is poorly understood. Studies during erythropoiesis have shown that iron bioavailability is coordinated with globin synthesis via bulk translation regulation. However, little is known about the control of translation during iron limitation in other tissues. Here, we investigated how iron depletion affects protein synthesis in human osteosarcoma U-2 OS cells. By adding an extracellular iron chelator, we observed that iron deficiency limits cell proliferation, induces autophagy, and decreases the global rate of protein synthesis. Analysis of specific molecular markers indicates that the inhibition of bulk translation upon iron limitation occurs through the eukaryotic initiation factor eIF2 alpha and mechanistic target of rapamycin (mTOR) pathways. In contrast to other environmental and nutritional stresses, iron depletion does not trigger the assembly of messenger ribonucleoprotein stress granules, which typically form upon polysome disassembly., We thank Dr. Maria Teresa Martinez-Pastor and the members of the Parker lab, especially Drs. Nina Ripin and Anthony Khong, for technical and scientific assistance. This research was supported by grant PID2020-116940RB-I00 and CEX2021-001189-S funded by MCIN/AEI/https://doi.org/10.13039/501100011033, and PRX21/00100 Fulbright Mobility Program Fellowship from Spanish Ministerio de Universidades to S.P.

[EN] Copper (Cu) deficiency compromises plant growth and limits crop productivity. Plants respond to Cu scarcity by activating the expression of several miRNAs, known as Cu-miRNAs, which degrade mRNAs from various cuproproteins to conserve Cu. Cu-miRNAs, like most plant miRNAs, associate with ARGONAUTE 1 (AGO1), the primary effector protein of miRNA-mediated gene silencing pathways, whose function is typically modulated by interacting proteins acting as cofactors. However, how AGO1 is regulated and functions under Cu deficiency remains unknown. Here, we searched for AGO1 interactors in Arabidopsis thaliana plants expressing a functional AGO1 protein tagged with the Twin-Strep-tag (TST) polypeptide, grown under Cu-sufficient or Cu-deficient conditions. TST-AGO1 complexes were affinity purified, and proteins were identified using tandem MS. Interestingly, the iron superoxide dismutase 1 (FeSOD1) encoded by FSD1 was enriched in TST-AGO1 complexes purified from plants grown under Cu deficiency. Moreover, fsd1-2 mutant plants showed reduced levels of AGO1 compared with wild-type plants under Cu sufficiency, while both Cu-miRNA-specific and general AGO1 target mRNAs accumulated to higher levels in fsd1-2 plants under both Cu-deficient and Cu-sufficient conditions compared with wild-type plants. These findings suggest that FeSOD1 is essential for proper AGO1 function, and that its superoxide dismutase activity, which mitigates oxidative stress, enhances AGO1 stability, particularly under Cu deficiency.; AGO1 is essential for Cu-deficiency responses but is sensitive to oxidative stress. FeSOD1 interacts with AGO1 and protects it from superoxide radical-induced degradation, thereby preserving miRNA-mediated gene silencing pathways., This work was supported by grants or fellowships from MCIN/AEI/10.13039/501100011033 (co-financed by the European Regional Development Fund) [RTI2018-095118-A-I00, PID2021-122186OB-I00 and RYC-2017-21648 to AC; PID2021-127671NB-I00 to GR; PID2020-116940RB-I00 to LP and APG; BIO2017-83184-R and PID2020-114691RB-I00 to JAD; PRE2019-088439 to AEC], and from the Generalitat Valenciana (CIPROM/2022/21 to GR and JAD).

[EN] Living organisms have developed finely regulated homeostatic networks to mitigate the effects of environmental fluctuations in transition metal micronutrients, including iron, zinc, and copper. In Saccharomyces cerevisiae, , the tandem zinc-finger protein Cth2 post-transcriptionally regulates gene expression under conditions of iron deficiency by controlling the levels of mRNAs that code for non-essential ferroproteins. The molecular mechanism involves Cth2 binding to AU-rich elements present in the 3 ' ' untranslated region of target mRNAs, negatively affecting their stability and translation. Arabidopsis thaliana has two TZF proteins homologous to yeast Cth2, C3H14 and C3H15, which participate in cell wall remodelling. The present work examines the expression of representative metal homeostasis genes with putative AREs in plants with altered levels of C3H14 and C3H15 grown under varying metal availabilities. The results suggest that C3H15 may act as a post-transcriptional plant modulator of metal adequacy, as evidenced by the expression of SPL7, , the main transcriptional regulator under copper deficiency, and PETE2, , which encodes plastocyanin. In contrast to S. cerevisiae, , the plant C3H15 affects copper and zinc homeostasis rather than iron. When grown under copper-deficient conditions, adult C3H15OE OE plants exhibit lower chlorophyll content and photosynthetic efficiency compared to control plants, suggesting accelerated senescence. Likewise, metal content in C3H15OE(OE) plants under copper deficiency shows altered mobilization of copper and zinc to seeds. These data suggest that the C3H15 protein plays a role in modulating both cell wall remodelling and metal homeostasis. The interaction between these processes may be the cause of altered metal translocation., We acknowledge Dr Patricia Casino for her help with the overlapping of the TZF domains, Dr Zhou for the C3H14, C3H15 overexpressing seeds and c3h14c3h15 (+/-) +/-) mutant and Marta Olmos for her technical support. A. Sanz obtained a sabbatical year grant (UV-PDI_SAB-156039). We acknowledge the SCSIE (Universitat de Valencia) for the ICP-MS service. This research was funded by the Spanish Ministry of Economy and Competitiveness "PID2020-116940RB-I00 and PID2023-148124OB-I00 funded by MCIN/AEI/10.13039/501100011033" and CIGE/2021/092 funded by Generalitat Valenciana.