Publications

The John Innes Centre Publications Repository contains details of all publications resulting from our researchers.

The repository also includes Open Access publications, which can be identified by the icons found on search results.

 Green open access publications are marked by the PDF icon. Click on the publication title, or the PDF icon, and read a pre-print PDF version of the publication.  Gold open access publications have the gold open padlock icon. You can read the full version of these papers on the publishing journal’s website without a subscription. 

The creation of this publications repository was funded by BBSRC.

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Connorton J. M., Jones E. R., Rodriguez-Ramiro I., Fairweather-Tait S., Uauy C., Balk J. (2017)

Vacuolar Iron Transporter TaVIT2 transports Fe and Mn and is effective for biofortification.

Plant Physiology (TBC) TBC

Publisher's version: 10.1104/pp.17.00672

ID: 56920

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Abstract

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. 

Connorton J. M., Balk J., Rodríguez-Celma J. (2017)

Iron homeostasis in plants - a brief overview.

Metallomics : integrated biometal science (TBC) TBC

Publisher's version: 10.1039/c7mt00136c

ID: 56921

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Abstract

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.

Bastow E. L., Bych K., Crack J. C., Le Brun N., Balk J. (2016)

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: 55224

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Abstract

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.

Schaedler T. A., Faust B., Shintre C. A., Carpenter E. P., Srinivasan V., van Veen H. W., Balk J. (2015)

Structures and functions of mitochondrial ABC transporters.

Biochemical Society Transactions (43) 943-51

Publisher's version: 10.1042/BST20150118

ID: 52083

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Abstract

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.

Ozer H. K., Dlouhy A. C., Thornton J. D., Hu J., Liu Y., Barycki J. J., Balk J., Outten C. E. (2015)

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: 51923

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Abstract

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.

Knuesting J., Riondet C., Maria C., Kruse I., Bécuwe N., König N., Berndt C., Tourrette S., Guilleminot-Montoya J., Herrero E., Gaymard F., Balk J., Belli G., Scheibe R., Reichheld J. P., Rouhier N., Rey P. (2015)

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: 49699

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Abstract

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.

Hooks M. A., Allwood J. W., Harrison J. K., Kopka J., Erban A., Goodacre R., Balk J. (2014)

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: 48259

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Abstract

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.

Schaedler T. A., Thornton J. D., Kruse I., Schwarzländer M., Meyer A. J., van Veen H. W., Balk J. (2014)

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: 48234

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Abstract

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.

Abstract

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.

Balk J., Schaedler T. A. (2014)

Iron Cofactor Assembly in Plants.

Annual Review of Plant Biology (TBC) TBC

Publisher's version: 10.1146/annurev-arplant-050213-035759

ID: 46415

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Abstract

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.

Wydro M. M., Sharma P., Foster J. M., Bych K., Meyer E. H., Balk J. (2013)

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: 44044

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Abstract

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.

Abstract

None

Bernard D. G., Netz D. J., Lagny T. J., Pierik A. J., Balk J. (2013)

Requirements of the cytosolic iron-sulfur cluster assembly pathway in Arabidopsis.

Philosophical transactions of the Royal Society London Series B Biological Sciences (368) 20120259

Publisher's version: 10.1098/rstb.2012.0259

ID: 43580

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Birke H., Haas F. H., De Kok L. J., Balk J., Wirtz M., Hell R. (2012)

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: 43576

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