The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.
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The creation of this publications repository was funded by BBSRC.
Pathogenic mutations in NUBPL affect complex I activity and cold tolerance in the yeast model Yarrowia lipolytica
Human Molecular Genetics
Publisher's version: 10.1093/hmg/ddy247
ID: 59404read more
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, encoding 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. Leu102Pro destabilized the Ind1 protein, leading to a null-mutant phenotype. Asp103Tyr, Leu191Phe and Gly285Cys affected complex I assembly to varying degrees, whereas Gly136Asp substitution in Ind1 did not impact on complex I levels nor 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 Ind1 Asp103Tyr 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 Gly285Cys variant was able to support complex I assembly at 28°C, but not at 10°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.
Plant Physiology (TBC) TBC
Publisher's version: 10.1101/306894
ID: 58722read more
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.
Scientific Reports (8) 6865
Publisher's version: 10.1038/s41598-018-25130-3
ID: 58455read more
Pea seeds are widely consumed in their immature form, known as garden peas and petit pois, mostly after preservation by freezing or canning. Mature dry peas are rich in iron in the form of ferritin, but little is known about the content, form or bioavailability of iron in immature stages of seed development. Using specific antibodies and in-gel iron staining, we show that ferritin loaded with iron accumulated gradually during seed development. Immunolocalization and high-resolution secondary ion mass spectrometry (NanoSIMS) revealed that iron-loaded ferritin was located at the surface of starch-containing plastids. Standard cooking procedures destabilized monomeric ferritin and the iron-loaded form. Iron uptake studies using Caco‑2 cells showed that the iron in microwaved immature peas was more bioavailable than in boiled mature peas, despite similar levels of soluble iron in the digestates. By manipulating the levels of phytic acid in the digestates we demonstrate that phytic acid is the main inhibitor of iron uptake from mature peas in vitro. Taken together, our data show that immature peas and mature dry peas contain similar levels of ferritin-iron, which is destabilized during cooking. However, iron from immature peas is more bioavailable because of lower phytic acid levels compared to mature peas.
Biochemical Journal (475) 495-509
Publisher's version: 10.1042/BCJ20170559
ID: 57905read more
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.
Plant Physiology (TBC) TBC
Publisher's version: 10.1104/pp.17.00672
ID: 56920read more
Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular iron deficiency anaemia is a major global health issue, but the iron content of staple crops such as wheat is difficult to change because of genetic complexity and homeostasis mechanisms. To identify target genes for biofortification of wheat (Triticum aestivum), we functionally characterized homologs of the Vacuolar Iron Transporter (VIT). The wheat genome contains two VIT paralogs, TaVIT1 and TaVIT2, which have different expression patterns, but are both low in the endosperm. TaVIT2, but not TaVIT1, was able to rescue growth of a yeast mutant lacking the vacuolar iron transporter. TaVIT2 also complemented a manganese transporter mutant, but not a vacuolar zinc transporter mutant. 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 legal fortification levels in countries such as the UK. The anti-nutrient phytate was not increased and the iron in the white flour fraction was bioavailable in-vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach impacted minimally on plant growth and was also effective in barley. Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue bypassing existing homeostatic mechanisms. Our results show that by enhancing vacuolar iron transport in the endosperm, this essential micronutrient accumulated in this tissue bypassing existing homeostatic mechanisms.
Metallomics : integrated biometal science (TBC) TBC
Publisher's version: 10.1039/c7mt00136c
ID: 56921read more
Iron plays a crucial role in biochemistry and is an essential micronutrient for plants and humans alike. Although plentiful in the Earth's crust it is not usually found in a form readily accessible for plants to use. They must therefore sense and interact with their environment, and have evolved two different molecular strategies to take up iron in the root. Once inside, iron is complexed with chelators and distributed to sink tissues where it is used predominantly in the production of enzyme cofactors or components of electron transport chains. The processes of iron uptake, distribution and metabolism are overseen by tight regulatory mechanisms, at the transcriptional and post-transcriptional level, to avoid iron concentrations building to toxic excess. Iron is also loaded into seeds, where it is stored in vacuoles or in ferritin. This is important for human nutrition as seeds form the edible parts of many crop species. As such, increasing iron in seeds and other tissues is a major goal for biofortification efforts by both traditional breeding and biotechnological approaches.
NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulfur proteins in Arabidopsis thaliana.
Plant Journal (89) 590-600
Publisher's version: 10.1111/tpj.13409
ID: 55224read more
Proteins of the cytosolic pathway for iron-sulfur (FeS) cluster assembly are conserved, except that plants lack a gene for CFD1 (Cytosolic FeS cluster Deficient 1). This poses the question of how NBP35 (Nucleotide-Binding Protein 35 kDa), the heteromeric partner of CFD1 in metazoa, functions on its own in plants. Firstly, we created viable mutant alleles of NBP35 in Arabidopsis to overcome embryo lethality of previously reported knockout mutations. RNAi knockdown lines with less than 30% NBP35 protein surprisingly showed no developmental or biochemical differences to wild type. Substitution of Cys14 to Ala, which destabilized the N-terminal Fe4S4 cluster in vitro, caused mild growth defects and a significant decrease in the activity of cytosolic FeS enzymes such as aconitase and aldehyde oxidases. The DNA glycosylase ROS1 was only partially decreased in activity and xanthine dehydrogenase not at all. Plants with strongly depleted NBP35 protein in combination with Cys14 to Ala substitution had distorted leaf development and decreased FeS enzyme activities. To find protein interaction partners of NBP35, a yeast two-hybrid screen was carried out which identified NBP35 and DRE2 (Derepressed for Ribosomal protein S14 Expression). NBP35 is known to form a dimer, and DRE2 acts upstream in the cytosolic FeS protein assembly pathway. The NBP35-DRE2 interaction was not disrupted by Cys14 to Ala substitution. Our results show that NBP35 has a function in the maturation of FeS proteins that is conserved in plants, and is closely allied to the function of DRE2.
Biochemical Society Transactions (43) 943-51
Publisher's version: 10.1042/BST20150118
ID: 52083read more
A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters.
Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System.
Journal of Biological Chemistry (290) 2782927840
Publisher's version: 10.1074/jbc.M115.682179
ID: 51923read more
The sulfhydryl oxidase Erv1 partners with the oxidoreductase Mia40 to import cysteine-rich proteins in the mitochondrial intermembrane space. In Saccharomyces cerevisiae, Erv1 has also been implicated in cytosolic Fe-S protein maturation and iron regulation. To investigate the connection between Erv1/Mia40-dependent mitochondrial protein import and cytosolic Fe-S cluster assembly, we measured Mia40 oxidation and Fe-S enzyme activities in several erv1 and mia40 mutants. While all the erv1 and mia40 mutants exhibited defects in Mia40 oxidation, only one erv1 mutant strain (erv1-1) had significantly decreased activities of cytosolic Fe-S enzymes. Further analysis of erv1-1 revealed that it had strongly decreased glutathione (GSH) levels, caused by an additional mutation in the gene encoding the glutathione biosynthesis enzyme glutamate cysteine ligase (GSH1). To address whether Erv1 or Mia40 plays a role in iron regulation, we measured iron-dependent expression of Aft1/2-regulated genes and mitochondrial iron accumulation in erv1 and mia40 strains. The only strain to exhibit iron misregulation is the GSH-deficient erv1-1 strain, which is rescued with addition of GSH. Together, these results confirm that GSH is critical for cytosolic Fe-S protein biogenesis and iron regulation, while ruling out significant roles for Erv1 or Mia40 in these pathways.
Arabidopsis glutaredoxin S17 and its partner, the nuclear factor Y subunit C11/negative cofactor 2a, contribute to maintenance of the shoot apical meristem under long-day photoperiod.
Plant Physiology (167) 1643-58
Publisher's version: 10.1104/pp.15.00049
ID: 49699read more
Glutaredoxins (GRXs) catalyze the reduction of protein disulfide bonds using glutathione as a reductant. Certain GRXs are able to transfer iron-sulfur clusters to other proteins. To investigate the function of Arabidopsis (Arabidopsis thaliana) GRXS17, we applied a strategy combining biochemical, genetic, and physiological approaches. GRXS17 was localized in the nucleus and cytosol, and its expression was elevated in the shoot meristems and reproductive tissues. Recombinant GRXS17 bound Fe2S2 clusters, a property likely contributing to its ability to complement the defects of a Baker's yeast (Saccharomyces cerevisiae) strain lacking the mitochondrial GRX5. However, a grxs17 knockout Arabidopsis mutant exhibited only a minor decrease in the activities of iron-sulfur enzymes, suggesting that its primary function is as a disulfide oxidoreductase. The grxS17 plants were sensitive to high temperatures and long-day photoperiods, resulting in elongated leaves, compromised shoot apical meristem, and delayed bolting. Both environmental conditions applied simultaneously led to a growth arrest. Using affinity chromatography and split-Yellow Fluorescent Protein methods, a nuclear transcriptional regulator, the Nuclear Factor Y Subunit C11/Negative Cofactor 2a (NF-YC11/NC2a), was identified as a GRXS17 interacting partner. A mutant deficient in NF-YC11/NC2a exhibited similar phenotypes to grxs17 in response to photoperiod. Therefore, we propose that GRXS17 interacts with NF-YC11/NC2a to relay a redox signal generated by the photoperiod to maintain meristem function.
Selective induction and subcellular distribution of ACONITASE 3 reveal the importance of cytosolic citrate metabolism during lipid mobilization in Arabidopsis.
Biochemical Journal (463) 309-317
Publisher's version: 10.1042/BJ20140430
ID: 48259read more
Arabidopsis thaliana has three genes that encode distinct aconitases (ACO), but little is known about the function of each isoenzyme during plant development. In newly emerged seedlings of Arabidopsis, transcript and protein levels for ACO3 were selectively induced to yield more than 80% of total aconitase activity. Characterization of knock-out mutants for each of the three ACOs suggests a major role for only ACO3 in citrate metabolism. The aco3 mutant displayed delayed early seedling growth, altered assimilation of [14C]acetate feeding, and elevated citrate levels, which were nearly 4-fold greater than in wild type, aco1 or aco2. However, both ACO1 and ACO2 are active in seedlings as shown by inhibition of aco3 growth by the toxin monofluoroacetate, and altered [14C]acetate assimilation and metabolite levels in aco1 and aco2. Relative levels of fumarate and malate differed between aco2 and aco3 indicating metabolically isolated pools of these metabolites in seedlings. Our inability to enrich ACO protein through mitochondria isolation, and the reduced cytosolic ACO activity of the iron-sulfur center assembly mutant atm3-1, indicated a cytosolic localization of ACO3 in 3 day-old seedlings. Subsequently, we determined that more than 90% of ACO3 was cytosolic. We conclude that ACO3 is cytosolic in young seedlings and functions in citrate catabolism consistent with the operation of the classic glyoxylate and not direct catabolism of citrate within mitochondria.
A conserved mitochondrial ATP-binding cassette transporter exports glutathione polysulfide for cytosolic metal cofactor assembly.
Journal of Biological Chemistry (289) 23264-23274
Publisher's version: 10.1074/jbc.M114.553438
ID: 48234read more
An ATP-binding cassette (ABC) transporter located in the inner mitochondrial membrane is involved in iron-sulfur cluster and molybdenum cofactor assembly in the cytosol, but the transported substrate is unknown. ATM3 (ABCB25) from Arabidopsis thaliana and its functional orthologue Atm1 from Saccharomyces cerevisiae were expressed in Lactococcus lactis, and studied in inside-out membrane vesicles and in purified form. Both proteins selectively transported glutathione disulfide (GSSG) but not reduced glutathione (GSH) in agreement with a 3-fold stimulation of ATPase activity by GSSG. By contrast, Fe2+ alone or in combination with glutathione did not stimulate ATPase activity. Arabidopsis atm3 mutants were hypersensitive to an inhibitor of glutathione biosynthesis, and accumulated GSSG in the mitochondria. The growth phenotype of atm3-1 was strongly enhanced by depletion of the mitochondria-localized, GSH-dependent persulfide oxygenase ETHE1, suggesting that the physiological substrate of ATM3 contains persulfide in addition to glutathione. Consistent with this idea, a transportomics approach using mass spectrometry showed that glutathione trisulfide (GS-S-SG) was transported by Atm1. We propose that mitochondria export glutathione polysulfide, containing glutathione and persulfide, for iron-sulfur cluster assembly in the cytosol.
Biofortification of wheat grain with iron and zinc: integrating novel genomic resources and knowledge from model crops
Frontiers in Plant Science (5) -
Publisher's version: 10.3389/fpls.2014.00053
ID: 46300read more
Wheat, like many other staple cereals, contains low levels of the essential micronutrients iron and zinc. Up to two billion people worldwide suffer from iron and zinc deficiencies, particularly in regions with predominantly cereal-based diets. Although wheat flour is commonly fortified during processing, an attractive and more sustainable solution is biofortification, which requires developing new varieties of wheat with inherently higher iron and zinc content in their grains. Until now most studies aimed at increasing iron and zinc content in wheat grains have focused on discovering natural variation in progenitor or related species. However, recent developments in genomics and transformation have led to a step change in targeted research on wheat at a molecular level. We discuss promising approaches to improve iron and zinc content in wheat using knowledge gained in model grasses. We explore how the latest resources developed in wheat, including sequenced genomes and mutant populations, can be exploited for biofortification. We also highlight the key research and practical challenges that remain in improving iron and zinc content in wheat.
Annual Review of Plant Biology (TBC) TBC
Publisher's version: 10.1146/annurev-arplant-050213-035759
ID: 46415read more
Iron is an essential element for all photosynthetic organisms. The biological use of this transition metal is as an enzyme cofactor, predominantly in electron transfer and catalysis. The main forms of iron cofactor are, in order of decreasing abundance, iron-sulfur clusters, heme, and di-iron or mononuclear iron, with a wide functional range. In plants and algae, iron-sulfur cluster assembly pathways of bacterial origin are localized in the mitochondria and plastids, where there is a high demand for these cofactors. A third iron-sulfur cluster assembly pathway is present in the cytosol that depends on the mitochondria but not on plastid assembly proteins. The biosynthesis of heme takes place mainly in the plastids. The importance of iron-sulfur cofactors beyond photosynthesis and respiration has become evident with recent discoveries of novel iron-sulfur proteins involved in epigenetics and DNA metabolism. In addition, increased understanding of intracellular iron trafficking is opening up research into how iron is distributed between iron cofactor assembly pathways and how this distribution is regulated. Expected final online publication date for the Annual Review of Plant Biology Volume 65 is April 29, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The Evolutionarily Conserved Iron-Sulfur Protein INDH1 Is Required for Complex I Assembly and Mitochondrial Translation in Arabidopsis.
Plant Cell (25) 4014-27
Publisher's version: 10.1105/tpc.113.117283
ID: 44044read more
The assembly of respiratory complexes is a multistep process, requiring coordinate expression of mitochondrial and nuclear genes and cofactor biosynthesis. We functionally characterized the iron-sulfur protein required for NADH dehydrogenase (INDH) in the model plant Arabidopsis thaliana. An indh knockout mutant lacked complex I but had low levels of a 650-kD assembly intermediate, similar to mutations in the homologous NUBPL (nucleotide binding protein-like) in Homo sapiens. However, heterozygous indh/+ mutants displayed unusual phenotypes during gametogenesis and resembled mutants in mitochondrial translation more than mutants in complex I. Gradually increased expression of INDH in indh knockout plants revealed a significant delay in reassembly of complex I, suggesting an indirect role for INDH in the assembly process. Depletion of INDH protein was associated with decreased (35)S-Met labeling of translation products in isolated mitochondria, whereas the steady state levels of several mitochondrial transcripts were increased. Mitochondrially encoded proteins were differentially affected, with near normal levels of cytochrome c oxidase subunit2 and Nad7 but little Nad6 protein in the indh mutant. These data suggest that INDH has a primary role in mitochondrial translation that underlies its role in complex I assembly.
Insights into the pathogenic character of a common NUBPL branch-site mutation associated with mitochondrial disease and complex I deficiency using a yeast model
Disease Models and Mechanisms (6) 1279-1284
Publisher's version: 10.1242/dmm.012682
ID: 43579read more
Philosophical transactions of the Royal Society London Series B Biological Sciences (368) 20120259
Publisher's version: 10.1098/rstb.2012.0259
ID: 43580read more
The DUF59 family gene AE7 acts in the cytosolic iron-sulfur cluster assembly pathway to maintain nuclear genome integrity in Arabidopsis.
Plant Cell (24) 4135-48
Publisher's version: 10.1105/tpc.112.102608
ID: 43577read more
Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana.
Biochemical Journal (445) 275-83
Publisher's version: 10.1042/BJ20120038
ID: 43576read more