Biology subjects

Balk, J.

Publications and source records attributed to Balk, J..

18 recordsLinked to original sources

bioRxiv2026

A conserved mechanism for regulation of mtDNA copy number in eukaryotes

Mitochondrial mass and mitochondrial DNA (mtDNA) copy number are coupled to metabolic demand at the cellular, tissue and organismal level, however, the molecular basis for homeostatic regulation of mtDNA is not understood. Here we show that mitochondria and mtDNA copy number are regulated by compartmentalisation of iron-sulfur (Fe-S) clusters, glutathione and cysteine, a mechanism we exemplify in model systems ranging from plants to human cells. Using genome-wide CRISPR screens we discovered that the mitochondrial ABC-family transporter, ABCB7, is a negative regulator of mtDNA copy number. Partial silencing of ABCB7 in human cells increased mtDNA 2-3 fold, enhancing mitochondrial mass and function. ABCB7 silencing compelled co-incident mitochondrial accumulation and cytosolic depletion of Fe-S clusters, simultaneously engaging the cellular iron starvation response and stabilising the mitochondrial glutathione transporter, SLC25A39. Transport of glutathione from the cytosol into mitochondria was co-incident with mitochondrial cysteine accumulation and cytosolic cysteine depletion, which was necessary and sufficient to increase mtDNA copy number in an integrated stress response-dependent fashion, with induction of PGC1{beta} and ERR. Silencing or partial loss of function mutations in the ABCB7 homologs of D.melanogaster, S.cerevisiae and A. thaliana elicited similar increases of mtDNA within these organisms. These data reveal a fundamental metabolic logic coupling compartmentalisation of redox co-factors to organellar genome content; a conserved axis across eukaryotes that pre-dates several elements of the mtDNA replication machinery.

cell biology↗

bioRxiv2024

Iron and redox sensing in plants: properties of the hemerythrin-like domains of Arabidopsis BRUTUS and BRUTUS-LIKE2 proteins

Iron uptake in plants is negatively regulated by highly conserved hemerythrin (Hr) E3 ubiquitin ligases exemplified by Arabidopsis thaliana BRUTUS (BTS). Physiological studies suggest these are the elusive plant iron sensors, but biochemical evidence is lacking. Here we demonstrate that the N-terminal domains of BTS and BTS-LIKE2 (BTSL2) respectively bind three and two diiron centres within three closely packed Hr-like subdomains. The centres can be reversibly oxidized by O2 and H2O2, resulting in a di-Fe3+ form that is non-labile. In the reduced state, a proportion of the iron becomes labile, based on accessibility to Fe2+ chelators and reconstitution experiments, consistent with dynamic iron binding. Impaired iron binding and altered redox properties in the BTS dgl variant correlate with diminished capacity to suppress the downstream signalling cascade. These data provide the biochemical foundation for a mechanistic model of how BTS/Ls function as iron sensors that are unique to the plant kingdom.

biochemistry↗

bioRxiv2024

Identification of significant genome-wide associations and QTL underlying variation in seed protein composition in pea (Pisum sativum L.)

Pea seeds are a valuable source of plant proteins for human and animal nutrition and have various industrial applications. The relative abundance of different seed storage proteins affects protein quality, including digestibility and functional properties of protein extracts. Thus, understanding the genetic basis of seed protein composition is crucial to enhance protein quality and nutritional value through breeding. In this study, we employed two complementary approaches, Genome-Wide Association Study (GWAS) and Quantitative Trait Locus (QTL) mapping, to identify genetic loci underlying seed protein composition in pea. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the seed proteins, and their relative abundance was quantified using densitometric analysis. For GWAS, we analysed a diverse panel of 209 accessions genotyped with an 84,691 SNP array and identified genetic loci significantly associated with globulins, such as convicilin, vicilin, legumins; and non-globulins, including lipoxygenase, late embryogenesis abundant protein (LEA), and annexin-like protein. Additionally, using QTL mapping with 96 recombinant inbred lines (RILs), we mapped 11 QTL, including five that overlapped with regions identified by GWAS for the same proteins. Within these regions, we identified structural genes for seed proteins and other genes with predicted functions in protein biosynthesis, trafficking, and modification. This comprehensive genetic mapping study serves as a foundation for future breeding efforts to improve protein quality in pea and other legumes.

genetics↗

bioRxiv2023

Genetic basis of historical pea mutants that hyper-accumulate iron

The Pisum sativum (pea) mutants degenerate leaves (dgl) and bronze (brz) accumulate large amounts of iron in leaves. First described several decades ago, the two mutants have provided important insights into iron homeostasis in plants but the underlying mutations have remained unknown. Using exome sequencing we identified an in-frame deletion associated with dgl in a BRUTUS homologue. The deletion is absent from wild type and the original parent line. BRUTUS belongs to a small family of E3 ubiquitin ligases acting as negative regulators of iron uptake in plants. The brz mutation was previously mapped to chromosome 4, and superimposing this region to the pea genome sequence uncovered a mutation in OPT3, encoding an oligopeptide transporter with a plant-specific role in metal transport. The causal nature of the mutations was confirmed by additional genetic analyses. Identification of the mutated genes rationalises many of the previously described phenotypes and provides new insights into shoot-to-root signalling of iron deficiency. Furthermore, the non-lethal mutations in these essential genes suggest new strategies for biofortification of crops with iron. Significance statementTwo iron-accumulating pea mutants first described more than 30 years ago have greatly contributed to our understanding of iron homeostasis in plants, but the mutations were never identified. Here we show that the phenotypes are caused by mutations in the BRUTUS and OPT3 genes and how this leads to specific defects in iron signalling and leaf development.

plant biology↗

bioRxiv2022

The BRUTUS-LIKE E3 ligases BTSL1 and BTSL2 fine-tune transcriptional responses to balance Fe and Zn homeostasis

The mineral micronutrients zinc (Zn) and iron (Fe) are essential for plant growth and human nutrition, but interactions between the homeostatic networks of these two elements are not fully understood. Here we show that loss-of-function of BTSL1 and BTSL2, which encode partially redundant E3 ubiquitin ligases that negatively regulate Fe uptake, confers tolerance to Zn excess in Arabidopsis thaliana. Double btsl1 btsl2 mutant seedlings grown on high Zn medium accumulated similar amounts of Zn in roots and shoots as the wild type, but suppressed the accumulation of excess Fe in roots. RNA-seq analysis showed that roots of mutant seedlings had relatively higher expression of genes involved in Fe uptake (IRT1, FRO2, NAS) and in Zn storage (MTP3, ZIF1). Surprisingly, mutant shoots did not show the transcriptional Fe-deficiency response which is normally induced by Zn excess. Split-root experiments suggested that within roots the BTSL proteins act locally and downstream of systemic Fe deficiency signals. Together, our data show that constitutive low-level induction of the Fe-deficiency response protects btsl1 btsl2 mutants from Zn toxicity. We propose that BTSL protein function is disadvantageous in situations of external Zn and Fe imbalances, and formulate a general model for Zn-Fe interactions in plants. HighlightMutation of two E3 ligases that suppress iron uptake in roots also confers tolerance to zinc toxicity, identifying a regulatory point of interaction between iron and zinc homeostasis.

plant biology↗

bioRxiv2022

Asymmetric expression of homoeologous genes in wheat roots modulates the early phase of iron-deficiency signalling

Iron (Fe) limitation leads to dramatic changes in gene expression in plants, to induce iron uptake and mobilization, while at the same time restrict Fe-dependent metabolism and growth. Although transcriptional changes in response to Fe deficiency have recently been charted in wheat, this was performed at a stage when photosynthesis and growth were severely impacted, confounding primary and secondary responses. Here, we specifically uncover the transcriptional landscape of wheat roots during the early stages of the Fe deficiency response (4 and 8 days) and after Fe resupply. Root growth was significantly inhibited at day 4, but chlorosis only became apparent on day 8. The number of differentially expressed genes increased from 1386 on day 4 to 3538 on day 8, with an overlap of 2006 genes. Genes with dynamic changes in expression patterns include membrane transporters and transcription factors shown to be involved in Fe homeostasis in other plant species. Comparative analysis of the Fe deficiency response at 4, 8 and 20 days identified a core set of Fe-regulated genes. Analysis of the expression of homoeologs suggests an increase in induction bias at 8 days compared to 4 days particularly, A genome contributing high at 4 days and the A+D genomes at 8 days. Overall, our work will contribute towards fundamental knowledge of the Fe signalling networks in wheat and point to the interplay of the three sub-genomes in this hexaploid species to fine tune the transcriptional response.

plant biology↗

bioRxiv2022

RAREFAN: a webservice to identify REPINs and RAYTs in bacterial genomes

Compared to eukaryotes, repetitive sequences are rare in bacterial genomes and usually do not persist for long. Yet, there is at least one class of persistent prokaryotic mobile genetic elements: REPINs. REPINs are non-autonomous transposable elements replicated by single-copy transposases called RAYTs. REPIN-RAYT systems are mostly vertically inherited and have persisted in individual bacterial lineages for millions of years. Discovering and analyzing REPIN populations and their corresponding RAYT transposases in bacterial species can be rather laborious, hampering progress in understanding REPIN-RAYT biology and evolution. Here we present RAREFAN, a webservice that identifies REPIN populations and their corresponding RAYT transposase in a given set of bacterial genomes. We demonstrate RAREFANs capabilities by analyzing a set of 49 Stenotrophomonas maltophilia genomes, containing nine different REPIN-RAYT systems. We guide the reader through the process of identifying and analyzing REPIN-RAYT systems across S. maltophilia, highlighting erroneous associations between REPIN and RAYTs, and providing solutions on how to find correct associations. RAREFAN enables rapid, large-scale detection of REPINs and RAYTs, and provides insight into the fascinating world of intragenomic sequence populations in bacterial genomes. RAREFAN is available at http://rarefan.evolbio.mpg.de.

bioinformatics↗

bioRxiv2022

A two-gene strategy increases the iron and zinc concentration of wheat flour and improves mineral bioaccesibility for human nutrition.

Dietary deficiencies of iron and zinc cause human malnutrition globally, which can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success so far due to relatively low genetic variation. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat vacuolar iron transporter gene TaVIT2-D with constitutive expression of the rice nicotianamine synthase gene OsNAS2 has the potential to dramatically improve mineral micronutrient intake from wheat products. In two distinct bread wheat cultivars, we show that the VIT-NAS construct led to a two-fold increase in zinc to [~]50 {micro}g g-1 in wholemeal flour and a two-fold increase in both zinc and iron in hand-milled white flour. In highly pure, roller-milled white flour, the concentration of iron was enhanced three-fold to [~]25 {micro}g g-1. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines was associated with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. We conclude that the effects of each gene cassette are additive in altering the total concentration and distribution of iron and zinc in wheat grains. This demonstrates the potential of a transgenic approach to enhance the nutritional quality of wheat well beyond what is possible by breeding approaches in order to alleviate dietary mineral deficiencies.

plant biology↗

bioRxiv2021

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

plant biology↗

bioRxiv2021

Subcellular dynamics studies reveal how tissue-specific distribution patterns of iron are established in developing wheat grains

Understanding iron trafficking in plants is key to enhancing the nutritional quality of crops. Due to the difficulty of imaging iron in transit, little is known about iron translocation and distribution in developing seeds. A novel approach, combining 57Fe isotope labelling and NanoSIMS, was used to visualize iron translocation dynamics at the subcellular level in wheat grain, Triticum aestivum L. We were able to track the main route of iron from maternal tissues to the embryo through different cell types. Further evidence for this route was provided by genetically diverting iron into storage vacuoles, as confirmed by histological staining and TEM-EDS. Virtually all iron was found in intracellular bodies, indicating symplastic rather than apoplastic transport. Aleurone cells contained a new type of iron body, highly enriched in 57Fe, and most likely represents iron-nicotianamine being delivered to phytate globoids. Correlation with tissue-specific gene expression provides an updated model of iron homeostasis in cereal grains with relevance for future biofortification efforts.

plant biology↗

bioRxiv2020

Branched-chain amino acid catabolism depends on GRXS15 through mitochondrial lipoyl cofactor homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol and mitochondria. A single monothiol glutaredoxin (GRX) has been shown to be involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homologue GRXS15 has only partially been characterized. Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype. In an in-depth metabolic analysis, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis and the electron transport chain. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, 2-oxoglutarate, glycine and branched-chain amino acids (BCAAs). The most pronounced accumulation occurred in branched-chain -keto acids (BCKAs), the first degradation products resulting from deamination of BCAAs. In wild-type plants, pyruvate, 2-oxoglutarate, glycine and BCKAs are all metabolized through decarboxylation by four mitochondrial lipoyl cofactor-dependent dehydrogenase complexes. Because these enzyme complexes are very abundant and the biosynthesis of the lipoyl cofactor depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why lipoyl cofactor-dependent processes are most sensitive to restricted Fe-S supply in GRXS15 K83A mutants. One-sentence summaryDeficiency in GRXS15 restricts protein lipoylation and causes metabolic defects in lipoyl cofactor-dependent dehydrogenase complexes, with branched-chain amino acid catabolism as dominant bottleneck.

plant biology↗

bioRxiv2019

The Medicago truncatula Vacuolar Iron Transporter-Like proteins VTL4 and VTL8 deliver iron to endosymbiotic bacteria at different stages of the infection process

The symbiotic relationship between legumes and rhizobium bacteria in root nodules has a high demand for iron. The host plant is known to provide iron to developing bacteroids, but questions remain regarding which transporters are involved. Here, we characterize two Vacuolar Iron Transporter-Like (VTL) proteins in Medicago truncatula that are specifically expressed during nodule development. VTL4 is mostly expressed during early infection and the protein was localized to membranes and the infection thread. vtl4 mutants were delayed in nodule development. VTL8 is closely related to SEN1 in Lotus japonicus and expressed in the late stages of bacteroid differentiation. The VTL8 protein was localized to the symbiosome membrane. A mutant line lacking the tandemly-arranged VTL4 - VTL8 genes, named 13U, was unable to develop functional nodules and failed to fix nitrogen, which was restored by expression of VTL8 alone. Using a newly developed lux reporter to monitor iron status of the bacteroids, a slight decrease in luminescence was observed in vtl4 mutants and a strong decrease in the 13U mutant. Iron transport capability of VTL4 and VTL8 was shown by yeast complementation. Taken together, these data indicate that VTL-type transporters are the main route for delivering iron to symbiotic rhizobia.

plant biology↗

bioRxiv2019

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems

O_LIA highly negative glutathione redox potential (EGSH) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear.\nC_LIO_LIWe dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing.\nC_LIO_LIOur data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ABC transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability.\nC_LIO_LIWe identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.\nC_LI

plant biology↗

bioRxiv2018

Pathogenic mutations in NUBPL affect complex I activity and cold tolerance in the yeast model Yarrowia lipolytica

Complex I deficiency is a common cause of mitochondrial disease, resulting from mutations in genes encoding structural subunits, assembly factors or defects in mitochondrial gene expression. Advances in genetic diagnostics and sequencing have led to identification of several variants in NUBPL, an assembly factor of complex I, which are potentially pathogenic. To help assign pathogenicity and learn more about the function of NUBPL, amino acid substitutions were recreated in the homologous Ind1 protein of the yeast model Yarrowia lipolytica. L102P destabilized the Ind1 protein, leading to a null-mutant phenotype. D103Y, L191F and G285C affected complex I assembly to varying degrees, whereas the G138D variant did not impact on complex I levels or dNADH:ubiquinone activity. Blue-native PAGE and immunolabelling of the structural subunits NUBM and NUCM revealed that all Ind1 variants accumulated a Q-module intermediate of complex I. In the D103Y variant the matrix arm intermediate was virtually absent, indicating a dominant effect. Dysfunction of Ind1, but not absence of complex I, rendered Y. lipolytica sensitive to cold. The Ind1 G285C variant was able to support complex I assembly at 28{degrees}C, but not at 10{degrees}C. Our results indicate that Ind1 is required for progression of assembly from the Q module to the full matrix arm. Cold sensitivity could be developed as a phenotype assay to demonstrate pathogenicity of NUBPL mutations and other complex I defects.

biochemistry↗

bioRxiv2018

Vacuolar iron stores gated by NRAMP3 and NRAMP4 are the primary source of iron in germinating seeds

During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100 and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1, SAPX), Fe homeostasis (FER1, SUFB) and chlorophyll metabolism (HEMA1, NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4. Together these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids but not mitochondria.

plant biology↗

bioRxiv2017

Genetic dissection of cyclic pyranopterin monophosphate biosynthesis in plant mitochondria

Mitochondria play a key role in the biosynthesis of two metal cofactors, iron-sulfur (FeS) clusters and molybdenum cofactor (Moco). The two pathways intersect at several points, but a scarcity of mutants has hindered studies to better understand these links. We screened a collection of sirtinol-resistant Arabidopsis thaliana mutants for lines with decreased activities of cytosolic FeS enzymes and Moco enzymes. We identified a new mutant allele of ATM3, encoding the ATP-binding cassette Transporter of the Mitochondria 3 (systematic name ABCB25), confirming the previously reported role of ATM3 in both FeS cluster and Moco biosynthesis. We also identified a mutant allele in CNX2, Cofactor of Nitrate reductase and Xanthine dehydrogenase 2, encoding GTP 3',8-cyclase, the first step in Moco biosynthesis which is localized in the mitochondria. A single nucleotide polymorphism in cnx2-2 leads to substitution of Arg88 with Gln in the N-terminal FeS cluster-binding motif. cnx2-2 plants are small and chlorotic, with severely decreased Moco enzyme activities, but they performed better than a cnx2-1 knockout mutant, which could only survive with ammonia as nitrogen source. Measurement of cyclic pyranopterin monophosphate (cPMP) levels by LC-MS/MS showed that this Moco intermediate was below the limit of detection in both cnx2-1 and cnx2-2, and accumulated more than 10-fold in seedlings mutated in the downstream gene CNX5. Interestingly, atm3-1 mutants had less cPMP than wild type, correlating with previous reports of a similar decrease in nitrate reductase activity. Taken together, our data functionally characterise CNX2 and suggest that ATM3 is indirectly required for cPMP synthesis.

biochemistry↗

bioRxiv2017

BRUTUS-LIKE proteins moderate the transcriptional response to iron deficiency in roots

Iron is an essential micronutrient but in excess is toxic inside cells. Under iron deficiency, the expression of iron uptake genes is increased, but it is not known how the transcriptional response is controlled to avoid uptake of too much iron. The hemerythrin E3 ligases BRUTUS (BTS) and BTS-LIKE (BTSL) have previously been identified as negative regulators of the iron deficiency response. Our phylogenetic analysis indicated that BTSL proteins are present in dicotyledonous plants only and form a separate clade from BTS homologs. BTSL1 and BTSL2 in Arabidopsis thaliana are in a network with nearly all iron uptake genes, whereas BTS is in a shoot-specific network. BTSL1 and BTSL2 are expressed predominantly in the root epidermis and cortex, separate from BTS in the root stele, shoot and embryos. Mutant analysis identified BTSL2 as the dominant paralog of the otherwise redundant BTSL genes. The btsl double mutant had increased protein levels of FIT, the FER-like Iron deficiency-induced Transcription factor, and failed to switch off the transcriptional response upon iron resupply, leading to dramatic iron accumulation in roots and shoots. Protein interaction between the C-terminus of BTSL proteins and FIT indicate that FIT is a direct target for degradation. Taken together, our studies show that BTSL1 and BTSL2 control iron uptake in the epidermis and cortex, upstream of BTS in the vasculature and leaves.

plant biology↗

bioRxiv2017

Altering Expression Of A Vacuolar Iron Transporter Doubles Iron Content In White Wheat Flour

Iron deficiency anaemia is a major global health issue, which has prompted mandatory fortification of cereal products with iron salts or elemental iron in many countries around the world. Rather than post-harvest fortification, biofortification - increasing the intrinsic nutritional quality of crops - is a more sustainable way of alleviating nutrient deficiencies. To identify target genes for biofortification of wheat (Triticum aestivum), we functionally characterized homologues of the Vacuolar Iron Transporter (VIT). The wheat genome contains two VIT paralogues, TaVIT1 and TaVIT2, which have different expression patterns, but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to transport iron in a yeast complementation assay. TaVIT2 also transported manganese but not zinc. By over-expressing TaVIT2 under the control of an endosperm-specific promoter, we achieved a 2-fold increase in iron in white flour fractions, exceeding minimum UK legal fortification levels. The highiron trait was consistent across independent lines and was stable in the next generation and in two different growth conditions. The single-gene approach impacted minimally on plant growth and was also effective in barley. The anti-nutrient phytate was not increased in white flour from the cisgenic wheat lines, suggesting that food products made from it could contribute to improved iron nutrition.

plant biology↗