Iron biofortification of staple crops - lessons and challenges in plant genetics

Plants are the ultimate source of iron in our diet, either directly as staple crops and vegetables or indirectly via animal fodder. Increasing the iron concentration of edible parts of plants, known as biofortification, is seen as a sustainable approach to alleviate iron deficiency which is a major global health issue. Advances in sequencing and gene technology are accelerating both forward and reverse genetic approaches. In this review we summarize recent progress in iron biofortification using conventional plant breeding or transgenics. Interestingly, some of the gene targets already used for transgenic approaches are also identified as genetic factors for high iron in genome-wide association studies. Several quantitative trait loci and transgenes increase both iron and zinc, due to overlap in transporters and chelators for these two mineral micronutrients. Research efforts are predominantly aimed at increasing the total concentration of iron but enhancing its bioavailability is also addressed. In particular, increased biosynthesis of the metal chelator nicotianamine increases iron (and zinc) levels and improves bioavailability. The achievements to date are very promising in being able to provide sufficient iron in diets with less reliance on meat to feed a growing world population

Iron homeostasis in plants - a brief overview

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

Absence of Complex I Is Associated with Diminished Respiratory Chain Function in European Mistletoe

Parasitism is a life history strategy found across all domains of life whereby nutrition is obtained from a host. It is often associated with reductive evolution of the genome, including loss of genes from the organellar genomes [1, 2]. In some unicellular parasites, the mitochondrial genome (mitogenome) has been lost entirely, with far-reaching consequences for the physiology of the organism [3, 4]. Recently, mitogenome sequences of several species of the hemiparasitic plant mistletoe (Viscum sp.) have been reported [5, 6], revealing a striking loss of genes not seen in any other multicellular eukaryotes. In particular, the nad genes encoding subunits of respiratory complex I are all absent and other protein-coding genes are also lost or highly diverged in sequence, raising the question what remains of the respiratory complexes and mitochondrial functions. Here we show that oxidative phosphorylation (OXPHOS) in European mistletoe, Viscum album, is highly diminished. Complex I activity and protein subunits of complex I could not be detected. The levels of complex IV and ATP synthase were at least 5-fold lower than in the non-parasitic model plant Arabidopsis thaliana, whereas alternative dehydrogenases and oxidases were higher in abundance. Carbon flux analysis indicates that cytosolic reactions including glycolysis are greater contributors to ATP synthesis than the mitochondrial tricarboxylic acid (TCA) cycle. Our results describe the extreme adjustments in mitochondrial functions of the first reported multicellular eukaryote without complex I

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

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

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 and SAPX), Fe homeostasis (FER1 and SUFB), and chlorophyll metabolism (HEMA1 and 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

Functional Characterization of the Eukaryotic Cysteine Desulfurase Nfs1p from Saccharomyces cerevisiae

Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation. The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus. Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein. First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate. Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS. Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins. Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition. Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol. The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol. Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs. Finally, we identified a highly conserved N-terminal {beta}-sheet of Nfs1p as a functionally essential part of the protein. The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed

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

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake

Arabidopsis BRUTUS-LIKE E3 ligases negatively regulate iron uptake by targeting transcription factor FIT for recycling

Organisms need to balance sufficient uptake of iron (Fe) with possible toxicity. In plant roots, a regulon of uptake genes is transcriptionally activated under Fe deficiency, but it is unknown how this response is inactivated when Fe becomes available. Here we describe the function of 2 partially redundant E3 ubiquitin ligases, BRUTUS-LIKE1 (BTSL1) and BTSL2, in Arabidopsis thaliana and provide evidence that they target the transcription factor FIT, a key regulator of Fe uptake, for degradation. The btsl double mutant failed to effectively down-regulate the transcription of genes controlled by FIT, and accumulated toxic levels of Fe in roots and leaves. The C-terminal domains of BTSL1 and BTSL2 exhibited E3 ligase activity, and interacted with FIT but not its dimeric partner bHLH39. The BTSL proteins were able to poly-ubiquitinate FIT in vitro and promote FIT degradation in vivo. Thus, posttranslational control of FIT is critical to prevent excess Fe uptake

Structures and functions of mitochondrial ABC transporters

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