Novel rice iron biofortification approaches using expression of ZmYS1 and OsTOM1 controlled by tissue-specific promoters.

Intrinsic improvement of iron (Fe) concentration in rice grains, called rice Fe biofortification, is a promising countermeasure against widespread human Fe deficiency. In this study, two novel rice Fe biofortification approaches are reported. The first approach (Y approach) involved the expression of maize YELLOW STRIPE 1 controlled by the HEAVY METAL ATPASE 2 promoter. The Y approach increased the polished grain Fe concentrations up to 4.8-fold compared with the non-transgenic (NT) line. The second approach (T approach) involved the expression of rice TRANSPORTER OF MUGINEIC ACID 1 controlled by the FERRIC REDUCTASE DEFECTIVE LIKE 1 promoter. The T approach increased the polished grain Fe concentrations by up to 3.2-fold. No synergistic increases in the polished grain Fe concentrations were observed when Y and T approaches were combined (YT approach). However, the polished grain Fe concentrations further increased by 5.1- to 9.3-fold compared with the NT line, when YT approach was combined with the endosperm-specific expression of FERRITIN (YTF approach), or when YTF approach was combined with the constitutive expression of NICOTIANAMINE SYNTHASE (YTFN approach). Total grain weight per plant in most Y, T, YT, and YTFN lines was comparable to that in the NT line, while it was significantly decreased in most YTF lines. The novel approaches reported in this study expand the portfolio of genetic engineering strategies that can be used for Fe biofortification in rice.

Selection of Suitable Reference Genes for qRT-PCR Gene Expression Studies in Rice.

With a widely established use of quantitative real-time PCR (qRT-PCR) for gene expression analysis, reliable and stable expression of reference genes is often discussed. Suitable reference genes should show less variation of expression across the target samples and allow for error minimization by normalization of qRT-PCR data. Therefore, selection of reliable reference genes is essential for accurate results and to support the conclusions drawn on expression levels of genes under study. In this chapter, we describe the workflow for selection and evaluation of reference genes in rice, including identification of candidate genes by using Genevestigator® and evaluation of expression stability using various algorithms. The ranking of the genes guides qRT-PCR performance and data analysis. This protocol used rice as an example but is not limited to rice, and could be applied to other species as well.

Delineating the future of iron biofortification studies in rice: challenges and future perspectives.

Iron (Fe) deficiency in humans is a widespread problem worldwide. Fe biofortification of rice (Oryza sativa) is a promising approach to address human Fe deficiency. Since its conceptualization, various biofortification strategies have been developed, some of which have resulted in significant increases in grain Fe concentration. However, there are still many aspects that have not yet been addressed in the studies to date. In this review, we first overview the important rice Fe biofortification strategies reported to date and the complications associated with them. Next, we highlight the key outstanding questions and hypotheses related to rice Fe biofortification. Finally, we make suggestions for the direction of future rice biofortification studies.

Multiplying the efficiency and impact of biofortification through metabolic engineering.

Ending all forms of hunger by 2030, as set forward in the UN-Sustainable Development Goal 2 (UN-SDG2), is a daunting but essential task, given the limited timeline ahead and the negative global health and socio-economic impact of hunger. Malnutrition or hidden hunger due to micronutrient deficiencies affects about one third of the world population and severely jeopardizes economic development. Staple crop biofortification through gene stacking, using a rational combination of conventional breeding and metabolic engineering strategies, should enable a leap forward within the coming decade. A number of specific actions and policy interventions are proposed to reach this goal.

Iron deficiency triggered transcriptome changes in bread wheat.

A series of complex transport, storage and regulation mechanisms control iron metabolism and thereby maintain iron homeostasis in plants. Despite several studies on iron deficiency responses in different plant species, these mechanisms remain unclear in the allohexaploid wheat, which is the most widely cultivated commercial crop. We used RNA sequencing to reveal transcriptomic changes in the wheat flag leaves and roots, when subjected to iron limited conditions. We identified 5969 and 2591 differentially expressed genes (DEGs) in the flag leaves and roots, respectively. Genes involved in the synthesis of iron ligands i.e., nicotianamine (NA) and deoxymugineic acid (DMA) were significantly up-regulated during iron deficiency. In total, 337 and 635 genes encoding transporters exhibited altered expression in roots and flag leaves, respectively. Several genes related to MAJOR FACILITATOR SUPERFAMILY (MFS), ATP-BINDING CASSETTE (ABC) transporter superfamily, NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family and OLIGOPEPTIDE TRANSPORTER (OPT) family were regulated, indicating their important roles in combating iron deficiency stress. Among the regulatory factors, the genes encoding for transcription factors of BASIC HELIX-LOOP-HELIX (bHLH) family were highly up-regulated in both roots and the flag leaves. The jasmonate biosynthesis pathway was significantly altered but with notable expression differences between roots and flag leaves. Homoeologs expression and induction bias analysis revealed subgenome specific differential expression. Our findings provide an integrated overview on regulated molecular processes in response to iron deficiency stress in wheat. This information could potentially serve as a guideline for breeding iron deficiency stress tolerant crops as well as for designing appropriate wheat iron biofortification strategies.

Genome Wide Analysis of the Transcriptional Profiles in Different Regions of the Developing Rice Grains.

BACKGROUND: Rice is an important food source for humans worldwide. Because of its nutritional and agricultural significance, a number of studies addressed various aspects of rice grain development and grain filling. Nevertheless, the molecular processes underlying grain filling and development, and in particular the contributions of different grain tissues to these processes, are not understood. MAIN TEXT: Using RNA-sequencing, we profiled gene expression activity in grain tissues comprised of cross cells (CC), the nucellar epidermis (NE), ovular vascular trace (OVT), endosperm (EN) and the aleurone layer (AL). These tissues were dissected using laser capture microdissection (LCM) at three distinct grain development stages. The mRNA expression datasets offer comprehensive and new insights into the gene expression patterns in different rice grain tissues and their contributions to grain development. Comparative analysis of the different tissues revealed their similar and/or unique functions, as well as the spatio-temporal regulation of common and tissue-specific genes. The expression patterns of genes encoding hormones and transporters indicate an important role of the OVT tissue in metabolite transport during grain development. Gene co-expression network prediction on OVT-specific genes identified several distinct and common development-specific transcription factors. Further analysis of enriched DNA sequence motifs proximal to OVT-specific genes revealed known and novel DNA sequence motifs relevant to rice grain development. CONCLUSION: Together, the dataset of gene expression in rice grain tissues is a novel and useful resource for further work to dissect the molecular and metabolic processes during rice grain development.

Potential Implications of Interactions between Fe and S on Cereal Fe Biofortification.

Iron (Fe) and sulfur (S) are two essential elements for plants, whose interrelation is indispensable for numerous physiological processes. In particular, Fe homeostasis in cereal species is profoundly connected to S nutrition because phytosiderophores, which are the metal chelators required for Fe uptake and translocation in cereals, are derived from a S-containing amino acid, methionine. To date, various biotechnological cereal Fe biofortification strategies involving modulation of genes underlying Fe homeostasis have been reported. Meanwhile, the resultant Fe-biofortified crops have been minimally characterized from the perspective of interaction between Fe and S, in spite of the significance of the crosstalk between the two elements in cereals. Here, we intend to highlight the relevance of Fe and S interrelation in cereal Fe homeostasis and illustrate the potential implications it has to offer for future cereal Fe biofortification studies.

Targeting intracellular transport combined with efficient uptake and storage significantly increases grain iron and zinc levels in rice.

Rice, a staple food for more than half of the world population, is an important target for iron and zinc biofortification. Current strategies mainly focus on the expression of genes for efficient uptake, long-distance transport and storage. Targeting intracellular iron mobilization to increase grain iron levels has not been reported. Vacuole is an important cell compartment for iron storage and the NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family of transporters export iron from vacuoles to cytosol when needed. We developed transgenic Nipponbare rice lines expressing AtNRAMP3 under the control of the UBIQUITIN or rice embryo/aleurone-specific 18-kDa Oleosin (Ole18) promoter together with NICOTIANAMINE SYNTHASE (AtNAS1) and FERRITIN (PvFER), or expressing only AtNRAMP3 and PvFER together. Iron and zinc were increased close to recommended levels in polished grains of the transformed lines, with maximum levels when AtNRAMP3, AtNAS1 and PvFER were expressed together (12.67 µg/g DW iron and 45.60 µg/g DW zinc in polished grains of line NFON16). Similar high iron and zinc levels were obtained in transgenic Indica IR64 lines expressing the AtNRAMP3, AtNAS1 and PvFER cassette (13.65 µg/g DW iron and 48.18 µg/g DW zinc in polished grains of line IR64_1), equalling more than 90% of the recommended iron increase in rice endosperm. Our results demonstrate that targeting intracellular iron stores in combination with iron and zinc transport and endosperm storage is an effective strategy for iron biofortification. The increases achieved in polished IR64 grains are of dietary relevance for human health and a valuable nutrition trait for breeding programmes.

Molecular processes in iron and zinc homeostasis and their modulation for biofortification in rice.

More than a billion people suffer from iron or zinc deficiencies globally. Rice (Oryza sativa L.) iron and zinc biofortification; i.e., intrinsic iron and zinc enrichment of rice grains, is considered the most effective way to tackle these deficiencies. However, rice iron biofortification, by means of conventional breeding, proves difficult due to lack of sufficient genetic variation. Meanwhile, genetic engineering has led to a significant increase in the iron concentration along with zinc concentration in rice grains. The design of impactful genetic engineering biofortification strategies relies upon vast scientific knowledge of precise functions of different genes involved in iron and zinc uptake, translocation and storage. In this review, we present an overview of molecular processes controlling iron and zinc homeostasis in rice. Further, the genetic engineering approaches adopted so far to increase the iron and zinc concentrations in polished rice grains are discussed in detail, highlighting the limitations and/or success of individual strategies. Recent insight suggests that a few genetic engineering strategies are commonly utilized for elevating iron and zinc concentrations in different genetic backgrounds, and thus, it is of great importance to accumulate scientific evidence for diverse genetic engineering strategies to expand the pool of options for biofortifying farmer-preferred cultivars.

Facilitated citrate-dependent iron translocation increases rice endosperm iron and zinc concentrations.

Iron deficiency affects one third of the world population. Most iron biofortification strategies have focused on genes involved in iron uptake and storage but facilitating internal long-distance iron translocation has been understudied for increasing grain iron concentrations. Citrate is a primary iron chelator, and the transporter FERRIC REDUCTASE DEFECTIVE 3 (FRD3) loads citrate into the xylem. We have expressed AtFRD3 in combination with AtNAS1 (NICOTIANAMINE SYNTHASE 1) and PvFER (FERRITIN) or with PvFER alone to facilitate long-distance iron transport together with efficient iron uptake and storage in the rice endosperm. The citrate and iron concentrations in the xylem sap of transgenic plants increased two-fold compared to control plants. Iron and zinc levels increased significantly in polished and unpolished rice grains to more than 70% of the recommended estimated average requirement (EAR) for iron and 140% of the recommended EAR for zinc in polished rice grains. Furthermore, the transformed lines showed normal phenotypic growth, were tolerant to iron deficiency and aluminum toxicity, and had grain cadmium levels similar to control plants. Together, our results demonstrate that deploying FRD for iron biofortification has no obvious anti-nutritive effects and should be considered as an effective strategy for reducing human iron deficiency anemia.

Single genetic locus improvement of iron, zinc and ß-carotene content in rice grains.

Nearly half of the world's population obtains its daily calories from rice grains, which lack or have insufficient levels of essential micronutrients. The deficiency of micronutrients vital for normal growth is a global health problem, and iron, zinc and vitamin A deficiencies are the most prevalent ones. We developed rice lines expressing Arabidopsis NICOTIANAMINE SYNTHASE 1 (AtNAS1), bean FERRITIN (PvFERRITIN), bacterial CAROTENE DESATURASE (CRTI) and maize PHYTOENE SYNTHASE (ZmPSY) in a single genetic locus in order to increase iron, zinc and ß-carotene content in the rice endosperm. NAS catalyzes the synthesis of nicotianamine (NA), which is a precursor of deoxymugeneic acid (DMA) iron and zinc chelators, and also chelate iron and zinc for long distance transport. FERRITIN provides efficient storage of up to 4500 iron ions. PSY catalyzes the conversion of GGDP to phytoene, and CRTI performs the function of desaturases required for the synthesis of ß-carotene from phytoene. All transgenic rice lines have significantly increased ß-carotene, iron, and zinc content in the polished rice grains. Our results establish a proof-of-concept for multi-nutrient enrichment of rice grains from a single genetic locus, thus offering a sustainable and effective approach to address different micronutrient deficiencies at once.

Enhanced Grain Iron Levels in Rice Expressing an IRON-REGULATED METAL TRANSPORTER, NICOTIANAMINE SYNTHASE, and FERRITIN Gene Cassette.

Micronutrient malnutrition is widespread, especially in poor populations across the globe, and iron deficiency anemia is one of the most prevalent forms of micronutrient deficiencies. Iron deficiency anemia has severe consequences for human health, working ability, and quality of life. Several interventions including iron supplementation and food fortification have been attempted and met with varied degrees of success. Rice, which is a staple food for over half of the world's population, is an important target crop for iron biofortification. The genetic variability of iron content in the rice germplasm is very narrow, and thus, conventional breeding has not been successful in developing high iron rice varieties. Therefore, genetic engineering approaches have targeted at increasing iron uptake, translocation, and storage in the rice endosperm. We previously reported that AtIRT1, when expressed together with AtNAS1 and PvFERRITIN (PvFER) in high-iron (NFP) rice, has a synergistic effect of further increasing the iron concentration of polished rice grains. We have now engineered rice expressing AtIRT1, AtNAS1, and PvFER as a single locus gene cassette and compared the resulting lines with transgenic lines expressing AtIRT1 and PvFER gene cassettes. We also evaluated the efficacies of the MsENOD12B and native AtIRT1 promoters for the expression of AtIRT1 in rice in both types of gene cassettes, and found the native AtIRT1 promoter to be a better choice for driving the AtIRT1 expression in our biofortification strategy. All the single insertion transgenic lines have significant increases of iron concentration, both in polished and unpolished grains, but the concerted expression of AtIRT1, AtNAS1, and PvFER resulted to be a more effective strategy in achieving the highest iron increases of up to 10.46 µg/g dry weight. Furthermore, the transformed high iron lines grew better under iron deficiency growth conditions and also have significantly increased grain zinc concentration. Together, these rice lines have nutritionally relevant increases in polished grain iron and zinc concentration necessary to support human health.

Iron biofortification in the 21st century: setting realistic targets, overcoming obstacles, and new strategies for healthy nutrition.

Plant-based foods offer a wide range of nutrients that are essential for human and animal health. Among these nutrients, iron stands out as one of the most important micronutrients. Increasing the iron content in many staple and non-staple plant foods continues to be a goal of many scientists around the world. However, the success of such initiatives has sometimes fallen short of their expected targets. In this review we highlight the most recent and promising results that have contributed to increasing the iron content in different crops. We also discuss methods that to date have been used to reach iron biofortification goals and new strategies that we believe are most promising for crop biofortification in the future. Plant anatomical, physiological and metabolic hurdles still need to be tackled for making progress on further increasing currently reached levels of micronutrient improvements. New strategies need to take into account growing environmental challenges that may constrain biofortification efforts.

Rice NICOTIANAMINE SYNTHASE 2 expression improves dietary iron and zinc levels in wheat.

KEY MESSAGE: Iron and zinc deficiencies negatively impact human health worldwide. We developed wheat lines that meet or exceed recommended dietary target levels for iron and zinc in the grains. These lines represent useful germplasm for breeding new wheat varieties that can reduce iron and zinc deficiency-associated health burdens in the affected populations. Micronutrient deficiencies, including iron and zinc deficiencies, have negative impacts on human health globally. Iron-deficiency; anemia affects nearly two billion people worldwide and is the cause of reduced cognitive development, fatigue and overall low productivity. Similarly, zinc deficiency causes stunted growth, decreased immunity and increased risk of respiratory infections. Biofortification of staple crops is a sustainable and effective approach to reduce the burden of health problems associated with micronutrient deficiencies. Here, we developed wheat lines expressing rice NICOTIANAMINE SYNTHASE 2 (OsNAS2) and bean FERRITIN (PvFERRITIN) as single genes as well as in combination. NAS catalyzes the biosynthesis of nicotianamine (NA), which is a precursor of the iron chelator deoxymugeneic acid (DMA) required for long distance iron translocation. FERRITIN is important for iron storage in plants because it can store up to 4500 iron ions. We obtained significant increases of iron and zinc content in wheat grains of plants expressing either OsNAS2 or PvFERRTIN, or both genes. In particular, wheat lines expressing OsNAS2 greatly surpass the HarvestPlus recommended target level of 30 % dietary estimated average requirement (EAR) for iron, and 40 % of EAR for zinc, with lines containing 93.1 µg/g of iron and 140.6 µg/g of zinc in the grains. These wheat lines with dietary significant levels of iron and zinc represent useful germplasm for breeding new wheat varieties that can reduce micronutrient deficiencies in affected populations.

Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib.

Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level.

NOD promoter-controlled AtIRT1 expression functions synergistically with NAS and FERRITIN genes to increase iron in rice grains.

Rice is a staple food for over half of the world's population, but it contains only low amounts of bioavailable micronutrients for human nutrition. Consequently, micronutrient deficiency is a widespread health problem among people who depend primarily on rice as their staple food. Iron deficiency anemia is one of the most serious forms of malnutrition. Biofortification of rice grains for increased iron content is an effective strategy to reduce iron deficiency. Unlike other grass species, rice takes up iron as Fe(II) via the IRON REGULATED TRANSPORTER (IRT) in addition to Fe(III)-phytosiderophore chelates. We expressed Arabidopsis IRT1 (AtIRT1) under control of the Medicago sativa EARLY NODULIN 12B promoter in our previously developed high-iron NFP rice lines expressing NICOTIANAMINE SYNTHASE (AtNAS1) and FERRITIN. Transgenic rice lines expressing AtIRT1 alone had significant increases in iron and combined with NAS and FERRITIN increased iron to 9.6 µg/g DW in the polished grains that is 2.2-fold higher as compared to NFP lines. The grains of AtIRT1 lines also accumulated more copper and zinc but not manganese. Our results demonstrate that the concerted expression of AtIRT1, AtNAS1 and PvFERRITIN synergistically increases iron in both polished and unpolished rice grains. AtIRT1 is therefore a valuable transporter for iron biofortification programs when used in combination with other genes encoding iron transporters and/or storage proteins.

Identification of novel alleles of the rice blast resistance gene Pi54.

Rice blast is one of the most devastating rice diseases and continuous resistance breeding is required to control the disease. The rice blast resistance gene Pi54 initially identified in an Indian cultivar confers broad-spectrum resistance in India. We explored the allelic diversity of the Pi54 gene among 885 Indian rice genotypes that were found resistant in our screening against field mixture of naturally existing M. oryzae strains as well as against five unique strains. These genotypes are also annotated as rice blast resistant in the International Rice Genebank database. Sequence-based allele mining was used to amplify and clone the Pi54 allelic variants. Nine new alleles of Pi54 were identified based on the nucleotide sequence comparison to the Pi54 reference sequence as well as to already known Pi54 alleles. DNA sequence analysis of the newly identified Pi54 alleles revealed several single polymorphic sites, three double deletions and an eight base pair deletion. A SNP-rich region was found between a tyrosine kinase phosphorylation site and the nucleotide binding site (NBS) domain. Together, the newly identified Pi54 alleles expand the allelic series and are candidates for rice blast resistance breeding programs.

Large scale germplasm screening for identification of novel rice blast resistance sources.

Rice is a major cereal crop that contributes significantly to global food security. Biotic stresses, including the rice blast fungus, cause severe yield losses that significantly impair rice production worldwide. The rapid genetic evolution of the fungus often overcomes the resistance conferred by major genes after a few years of intensive agricultural use. Therefore, resistance breeding requires continuous efforts of enriching the reservoir of resistance genes/alleles to effectively tackle the disease. Seed banks represent a rich stock of genetic diversity, however, they are still under-explored for identifying novel genes and/or their functional alleles. We conducted a large-scale screen for new rice blast resistance sources in 4246 geographically diverse rice accessions originating from 13 major rice-growing countries. The accessions were selected from a total collection of over 120,000 accessions based on their annotated rice blast resistance information in the International Rice Genebank. A two-step resistance screening protocol was used involving natural infection in a rice uniform blast nursery and subsequent artificial infections with five single rice blast isolates. The nursery-resistant accessions showed varied disease responses when infected with single isolates, suggesting the presence of diverse resistance genes/alleles in this accession collection. In addition, 289 accessions showed broad-spectrum resistance against all five single rice blast isolates. The selected resistant accessions were genotyped for the presence of the Pi2 resistance gene, thereby identifying potential accessions for isolation of allelic variants of this blast resistance gene. Together, the accession collection with broad spectrum and isolate specific blast resistance represent the core material for isolation of previously unknown blast resistance genes and/or their allelic variants that can be deployed in rice breeding programs.

Three-dimensional modeling and diversity analysis reveals distinct AVR recognition sites and evolutionary pathways in wild and domesticated wheat Pm3 R genes.

The Pm3 gene confers resistance against wheat powdery mildew. Studies of Pm3 diversity have shown that Pm3 alleles isolated from southern populations of wild emmer wheat located in Lebanon, Jordan, Israel, and Syria are more diverse and more distant from bread wheat alleles than alleles from the northern wild wheat populations located in Turkey, Iran, and Iraq. Therefore, southern populations from Israel were studied extensively to reveal novel Pm3 alleles that are absent from the cultivated gene pool. Candidate Pm3 genes were isolated via a polymerase chain reaction cloning approach. Known and newly identified Pm3 genes were subjected to variation analysis and polymorphic amino acid residues were superimposed on a three-dimensional (3D) model of PM3. The region of highest interspecies diversity between Triticum aestivum and T. dicoccoides lies in leucine-rich repeats (LRR) 19 to 24, whereas most intraspecies diversity in T. aestivum is located in LRR 25 to 28. Interestingly, these two regions are separated by one large LRR whose propensity for flexibility facilitates the conformation of the PM3 LRR domain into two differently structured models. The combination of evolutionary and protein 3D structure analysis revealed that Pm3 genes in wild and domesticated wheat show different evolutionary histories which might have been triggered through different interactions with the powdery mildew pathogen.