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Dealing with iron metabolism in rice: from breeding for stress tolerance to biofortification

Abstract

Iron is a well-known metal. Used by humankind since ancient times in many different ways, this element is present in all living organisms, where, unfortunately, it represents a two-way problem. Being an essential block in the composition of different proteins and metabolic pathways, iron is a vital component for animals and plants. That is why iron deficiency has a severe impact on the lives of different organisms, including humans, becoming a major concern, especially in developing countries where access to adequate nutrition is still difficult. On the other hand, this metal is also capable of causing damage when present in excess, becoming toxic to cells and affecting the whole organism. Because of its importance, iron absorption, transport and storage mechanisms have been extensively investigated in order to design alternatives that may solve this problem. As the understanding of the strategies that plants use to control iron homeostasis is an important step in the generation of improved plants that meet both human agricultural and nutritional needs, here we discuss some of the most important points about this topic.

Keywords:
iron toxicity; mineral malnutrition; Fe-enrichment; Quantitative Trait Loci

Introduction

Iron is the fourth most abundant element in the earth’s crust, where ferric iron (Fe3+) and ferrous iron (Fe2+) are the most common forms (Hori et al., 2015Hori T, Aoyagi T, Itoh H, Narihiro T, Oikawa A, Suzuki K, Ogata A, Friedrich MW, Conrad R and Kamagata Y (2015) Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments. Front Microbiol 6:386.). While Fe3+ is insoluble and its uptake is difficult, Fe2+ is soluble and readily available to plants. When the soil is aerated and in alkaline pH, Fe is oxidized as insoluble iron oxides, but in flooded soils, which are in anaerobic conditions, pH decreases and there is a reduction of Fe3+ to Fe2+ (Morrissey and Guerinot, 2009Morrissey J and Guerinot ML (2009) Iron uptake and transport in plants: The good, the bad, and the ionome. Chem Rev 109:4553-4567.). This event is responsible for the low availability of Fe in upland soils and for its high availability in flooded soils.

Fe is an essential micronutrient for both animals and plants. In mammals iron is part of the structure of a diversity of proteins (hemoglobin, myoglobin, cytochromes, flavoproteins, heme-flavoproteins, transferrin, lactoferrin, ferritin, hemosiderin, sulfur, non-heme enzymes) (Institute of Medicine, 2001Institute of Medicine (2001) Dietary reference intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium and Zinc. National Academy Press, Washington, DC, 773 p.). In plants, Fe serves as a component of many vital enzymes such as cytochromes of the electron transport chain, acting in photosynthesis and in the electron transfer (through Fe-S clusters), in respiration, and other important metabolic pathways (Briat and Lobreaux, 1997Briat JF and Lobreaux S (1997) Iron transport and storage in plants. Trends Plant Sci 2:187-193.; Kobayashi and Nishizawa, 2012Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.; Rout and Sahoo, 2015Rout GR and Sahoo S. (2015) Role of iron in plant growth and metabolism Rev Agric Sci 3:1-24.). It also participates in the Fenton reaction catalyzing the generation of hydroxyl radicals (OH), and reactive oxygen species (ROS) that can cause irreversible damage to the cell (Wu et al., 2014Wu L, Shhadi MY, Gregorio G, Matthus E, Becker M and Frei M (2014) Genetic and physiological analysis of tolerance to acute iron toxicity in rice. Rice 7:8.). Thus, Fe stress can be caused either by deficiency as well as by excess (Connolly and Guerinot, 2002Connolly EL and Guerinot M (2002) Iron stress in plants. Genome Biol 3:1024.1-1024.4.).

Iron deficiency can cause alterations in root morphology (Morrissey and Guerinot, 2009Morrissey J and Guerinot ML (2009) Iron uptake and transport in plants: The good, the bad, and the ionome. Chem Rev 109:4553-4567.; Giehl et al., 2012Giehl RFH, Lima JE and von Wirén N (2012) Localized iron supply triggers lateral root elongation in Arabidopsis by altering the AUX1-mediated auxin distribution Plant Cell 24:33-49.; Gruber et al., 2013Gruber BD, Giehl RFH, Friedel S and von Wirén N (2013) Plasticity of the Arabidopsis root system under nutrient deficiencies. Plant Physiol 163:161-179.) and chlorosis of young leaves, therefore reducing yield (Kobayashi and Nishizawa, 2014Kobayashi T and Nishizawa NK (2014) Iron sensors and signals in response to iron deficiency. Plant Sci 224:36-43.). To prevent the shortage of this element, plants have developed two different absorption strategies: strategy I, which is used by higher plants, except for members of the Poaceae family. In this strategy the enzyme H+ ATPase (AHA) mediates the release of hydrons from the roots to the rhizosphere, increasing the solubility of Fe3+, and the Phenolics Efflux Zero 1 (PEZ1) transports phenolics, such as protocatechuic acid, making it possible to take up and use apoplastic precipitated Fe (Ishimaru et al., 2011Ishimaru Y, Bashir K, Nakanishi H and Nishizawa NK (2011) The role of rice phenolics efflux transporter in solubilizing apoplasmic iron. Plant Signal Behav 6:1624-1626.; Rodríguez-Celma and Schmidt, 2013Rodríguez-Celma J and Schmidt W (2013) Reduction-based iron uptake revisited on the role of secreted iron-binding compounds. Plant Signal Behav 8:e26116.). A comparison between two model species, Arabidopsis and Medicago truncatula, showed further evidence that the production and secretion of phenolic compounds is critical for the uptake of iron from sources with low bioavailability, but dispensable under conditions where iron is readily available (Rodríguez-Celma et al., 2013Rodríguez-Celma J, Lin W-D, Fu G-M, Abadía J, López-Millán A-F and Schmidt W (2013) Mutually exclusive alterations in secondary metabolism are critical for the uptake of insoluble iron compounds by Arabidopsis and Medicago truncatula. Plant Physiol 162:1473-1485.; Rodríguez-Celma and Schmidt, 2013Rodríguez-Celma J and Schmidt W (2013) Reduction-based iron uptake revisited on the role of secreted iron-binding compounds. Plant Signal Behav 8:e26116.).

Also, in strategy I, Ferric Reductase Oxidase (FRO2) mediates the Fe3+ reduction to Fe2+, and Iron Regulated Transporter1 (IRT1) is responsible for Fe2+ absorption by the roots (Connolly and Guerinot, 2002Connolly EL and Guerinot M (2002) Iron stress in plants. Genome Biol 3:1024.1-1024.4.; Kobayashi and Nishizawa, 2012Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.).

Strategy II, which is specific of grasses, is based on biosynthesis and secretion of compounds called phytosiderophores (PS), which are results of the action of nicotianamine synthase (NAS), nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) (Shojima et al., 1990Shojima S, Nishizawa N-K, Fushiya S, Nozoe S, Irifune T and Mori S (1990) Biosynthesis of phytosiderophores. Plant Physiol 93:1497-1503.). TOM1/OsZIFL4, which belongs to the major facilitator superfamily (MFS) (Pao et al., 1998Pao SS, Paulsen IT and Saier Jr MH (1998) Major facilitator superfamily. Microbiol Mol Biol Rev 62:1-34.), is involved in siderophore export necessary in Fe acquisition (Furrer et al., 2002Furrer JL, Sanders DN, Hook-Barnard IG and McIntosh MA (2002) Export of the siderophore enterobactin in Escherichia coli: Involvement of a 43 kDa membrane exporter. Mol Microbiol 44:1225-1234.). These PSs can bind to Fe3+ forming the soluble complex Fe(III)-PS, and these complexes in the rhizosphere can be taken up into root cells through the action of YELLOW STRIPE-LIKE PROTEINS (YSLs) (Inoue et al., 2009Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori S and Nishizawa NK (2009) Rice OsYSL15 is an iron-regulated Iron(III)-Deoxymugineic Acid Transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings. J Biol Chem 284:3470-3479.; Lee et al., 2009aLee S, Chiecko JC, Kim SA, Walker EL, Lee Y, Guerinot ML and An G (2009a) Disruption of OsYSL15 leads to iron inefficiency in rice plants. Plant Physiol 150:786-800.; Nozoye et al., 2011Nozoye T, Nagasaka S, Kobayashi T, Takahashi M, Sato Y, Sato Y, Uozumi N, Nakanishi H and Nishizawa NK (2011) Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants. J Biol Chem 286:5446-5454.). In rice there are 18 YS1-like (OsYSL) genes, and OsYSL15 transports Fe(II)-PS and it is likely more relevant for Fe(III)-PS (Romheld and Marschner, 1986Romheld V and Marschner H (1986) Evidence for a specific uptake system for iron phytosiderophores in roots of grasses. Plant Physiol 80:175-180.; Curie et al., 2001Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF and Walker EL (2001) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409:346-349.; Inoue et al., 2009Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori S and Nishizawa NK (2009) Rice OsYSL15 is an iron-regulated Iron(III)-Deoxymugineic Acid Transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings. J Biol Chem 284:3470-3479.; Lee et al., 2009aLee S, Chiecko JC, Kim SA, Walker EL, Lee Y, Guerinot ML and An G (2009a) Disruption of OsYSL15 leads to iron inefficiency in rice plants. Plant Physiol 150:786-800.). Rice (Oryza sativa L.) uses strategy II, but is also able to absorb Fe2+ directly from the rhizosphere through IRT1 (Zaharieva and Römheld, 2000Zaharieva T and Römheld V (2000) Specific Fe2+ uptake system in strategy I plants inducible under Fe deficiency, J Plant Nutr 18:1733-1744.; Bughio et al., 2002Bughio N, Yamaguchi H, Nishizawa NK, Nakanishi H and Mori S (2002) Cloning an iron-regulated metal transporter from rice. J Exp Bot 53:1677-1682.; Ishimaru et al., 2006Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, et al. (2006) Rice plants take up iron as an Fe 3+ -phytosiderophore and as Fe 2+. Plant J 45:335-346.; Kobayashi and Nishizawa, 2014Kobayashi T and Nishizawa NK (2014) Iron sensors and signals in response to iron deficiency. Plant Sci 224:36-43.).

The high level of Fe2+ found in some flooded soils can be toxic to plants (Mongon et al., 2014Mongon J, Konnerup D, Colmer TD and Rerkasem B (2014) Responses of rice to Fe2+ in aerated and stagnant conditions: Growth, root porosity and radial oxygen loss barrier. Funct Plant Biol 41:922-929.). The toxicity caused by excessive Fe can occur directly and indirectly. The direct toxicity occurs when there is too much absorption and excessive accumulation of this element in tissues followed by the appearance of brown-dark spots in the leaves (leaf-bronzing) (Becker and Asch, 2005Becker M and Asch F (2005) Iron toxicity in rice - Conditions and management concepts. J Plant Nutr Soil Sci 168:558-573.; Morrissey and Guerinot, 2009Morrissey J and Guerinot ML (2009) Iron uptake and transport in plants: The good, the bad, and the ionome. Chem Rev 109:4553-4567.). The indirect damage is caused by the prevention of the uptake, transport and utilization of other nutrients (e.g.: P, K, Ca, Mg, Mn, and Zn) due to the iron plaque that forms when Fe3+ is deposited in the apoplast of rice roots (Sahrawat, 2004Sahrawat KL (2004) Managing iron toxicity in lowland rice: The role of tolerant genotypes and plant nutrients. In: Rice is life: Scientific perspectives for the 21st century. Proceedings of the World Rice Research Conference, Tsukuba, pp 452-455.; Zhang et al., 2014Zhang M, Pinson SRM, Tarpley L, Huang XY, Lahner B, Yakubova E, Baxter I, Guerinot ML and Salt DE (2014) Mapping and validation of quantitative trait loci associated with concentrations of 16 elements in unmilled rice grain. Theor Appl Genet 127:137-165.). Both situations affect plant growth, development and productivity, leading to significant yield losses. To adapt to this condition, rice plants have developed different mechanisms of tolerance (Type I, Type II and Type III) that are based on specific forms of use, exclusion and storage of iron. In Type I there is an oxidation and precipitation of Fe2+ on the root surface, while in Type II the storage occurs in a less reactive form, in ferritin protein. Type III mechanism is based on tolerance to the ROS formed in Fenton’s reaction (Wu et al., 2014Wu L, Shhadi MY, Gregorio G, Matthus E, Becker M and Frei M (2014) Genetic and physiological analysis of tolerance to acute iron toxicity in rice. Rice 7:8.). Another thing that can occur is the annulment of the absorbed Fe2+ by its storage in old or less active leaves or exclusion via symplast (Becker and Asch, 2005Becker M and Asch F (2005) Iron toxicity in rice - Conditions and management concepts. J Plant Nutr Soil Sci 168:558-573.).

Physiological disorders caused by Fe excess are common in cultivated rice in the regions of Africa, Asia and South America (Shahid et al., 2014Shahid M, Nayak AK, Shukla AK, Tripathi R, Kumar A, Raja R, Panda BB, Meher J, Bhattacharyya P and Dash D (2014) Mitigation of iron toxicity and Iron, Zinc, and Manganese nutrition of Wetland Rice Cultivars (Oryza sativa L.) grown in iron-toxic soil. CLEAN - Soil Air Water 42:1604-1609.). However, despite the high amount of Fe in the soil, which can even be toxic to the plant, little is accumulated in rice grains. In addition, the accumulation of iron in the grain occurs in the outermost layers being lost during the industrial processing (Doesthale et al., 1979Doesthale Y, Devara S, Rao S and Belavady B (1979) Effect of milling on mineral and trace element composition of raw and parboiled rice, J Sci Food Agric 30:40-46.; Sperotto et al., 2012Sperotto RA, Vasconcelos MW, Grusak MA and Fett JP (2012) Effects of different Fe supplies on mineral partitioning and remobilization during the reproductive development of rice (Oryza sativa L.). Rice 5:27.). Thus, rice contributes very little to meet the need of Fe intake in the human diet, not being an effective way of preventing anemia.

More than two billion people worldwide suffer from anemia, and more than 50% of these cases are caused by Fe deficiency (Arcanjo et al., 2013Arcanjo FPN, Santos PR and Arcanjo CPC (2013) Daily and weekly iron supplementations are effective in increasing hemoglobin and reducing anemia in infants. J Trop Pediatr 59:175-179.). The Fe-deficiency anemia (IDA) affects more dramatically the continents of Africa and Asia, where IDA is a major public health problem, prevalent in young women and children (Moretti et al., 2006Moretti D, Zimmermann MB, Muthayya S, Thankachan P, Lee TC, Kurpad AV and Hurrell RF (2006) Extruded rice fortified with micronized ground ferric pyrophosphate reduces iron deficiency in Indian schoolchildren: A double-blind randomized controlled trial1-3. Am J Clin Nutr 84:822-829.; Visser and Herselman, 2013Visser J and Herselman M (2013) Anaemia in South Africa: The past, the present and the future. S Afr J Clin Nutr 26:166-167.), since it is responsible for the death of almost one million individuals per year (Aung et al., 2013Aung MS, Masuda H, Kobayashi T, Nakanishi H, Yamakawa T and Nishizawa NK (2013) Iron biofortification of myanmar rice. Front Plant Sci 4:158.).

Biofortification is an interesting strategy to solve the problem of IDA, especially for people who cannot change their eating habits due to financial, cultural or religious issues. In this sense, not only increasing the amount of iron in grains, but also decreasing the content of inhibitors of Fe absorption commonly found in plants can improve the diets (Lucca et al., 2001Lucca P, Hurrell R and Potrykus I (2001) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet 102:392-397.; Raboy, 2002Raboy V (2002) Progress in breeding low phytate crops. J Nutr 503-505.; Schuler and Bauer, 2012Schuler M and Bauer P (2012) Strategies for iron biofortification of crop plants. In: Kapiris K (ed) Food Quality, InTech. Available from: http://www.intechopen.com/books/food-quality/strategies-for-iron-biofortification-of-crop-plants.
http://www.intechopen.com/books/food-qua...
). In addition, biofortification is a sustainable strategy. In this sense rice can be the ideal species for biofortification since it is a staple food that is especially important for developing countries, where IDA is even more severe. Also, rice is grown in flooded soils, where Fe availability is higher (Becker and Asch, 2005Becker M and Asch F (2005) Iron toxicity in rice - Conditions and management concepts. J Plant Nutr Soil Sci 168:558-573.), and has its mechanisms of absorption, translocation and homeostasis Fe better understood than most of the species (Masuda et al., 2012Masuda H, Ishimaru Y, Aung MS, Kobayashi T, Kakei Y, Takahashi M, Higuchi K, Nakanishi H and Nishizawa NK (2012) Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition. Sci Rep 2:1-7.).

Global rice production is 741 million tons at approximately 165 million hectares. Rice is not only the second most cultivated cereal in the world, with important social and economic function, but is also an ideal model for functional genomics studies in monocots (FAOSTAT, 2015FAOSTAT (2015) Food and Agriculture Organization of The United Nations - Statistics Division (FAOSTAT), http://faostat3.fao.org/download/Q/QC/E (August 31, 2015).
http://faostat3.fao.org/download/Q/QC/E...
; Yao et al., 2015Yao W, Li G, Zhao H, Wang G, Lian X and Xie W (2015) Exploring the rice dispensable genome using a metagenome-like assembly strategy. Genome Biol 16:187.). The availability of different rice genomes of different subspecies has enabled the study of many genes and metabolic pathways (Goff et al., 2002Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296:92-100.; IRGSP - International Rice Genome Sequencing Project, 2005IRGSP - International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436:793-800.).

Considering the importance of rice in nutrition and economy as well as the impact of iron deficiency and excess in the life of plants and animals, in this review we will discuss the highlights of the uptake pathways, translocation, homeostasis and Fe accumulation in the grain. Understanding these points is essential both to solve the problem of sensitivity to high levels of Fe as to allow Fe-biofortification.

Identifying regulatory pathways

According to the availability of Fe in the soil, plants have developed mechanisms to control and regulate the absorption, translocation and subcellular storage of this mineral. Classical studies associated with the emergence of modern and advanced tools of genomics, transcriptomics and proteomics have enabled in-depth understanding of homeostasis of Fe in plants (Kobayashi and Nishizawa, 2012Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.). The uptake of Fe occurs by using strategy I or reduction (non Poaceae), strategy II or chelation (Poaceae), and a combination of strategies I and II (rice) (Figure 1) (Ishimaru et al., 2006Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, et al. (2006) Rice plants take up iron as an Fe 3+ -phytosiderophore and as Fe 2+. Plant J 45:335-346.; Zhang et al., 2012Zhang Y, Xu YH, Yi HY and Gong JM (2012) Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. Plant J 72:400-410.; Yang et al., 2013Yang M, Zhang W, Dong H, Zhang Y, Lv K, Wang D and Lian X (2013) OsNRAMP3 is a vascular bundles-specific manganese transporter that is responsible for manganese distribution in rice. PLoS One 8:e83990.; Ricachenevsky and Sperotto, 2014Ricachenevsky FK and Sperotto RA (2014) There and back again, or always there? The evolution of rice combined strategy for Fe uptake. Front Plant Sci 5:189.; Finatto et al., 2015Finatto T, de Oliveira AC, Chaparro C, da Maia LC, Farias DR, Woyann LG, Mistura CC, Soares-Bresolin AP, Llauro C, Panaud O, et al. (2015) Abiotic stress and genome dynamics: Specific genes and transposable elements response to iron excess in rice. Rice 8:13.). The key genes involved in strategy I are AHA2 (protonation of the rhizosphere), FRO2 (reduction of Fe3+ to Fe2+), and IRT1 (Fe2+ transport into the root) (Kim and Guerinot, 2007Kim SA and Guerinot ML (2007) Mining iron: Iron uptake and transport in plants. FEBS Lett 581:2273-2280.; Hindt and Guerinot, 2012Hindt MN and Guerinot ML (2012) Getting a sense for signals: Regulation of the plant iron deficiency response. Biochim Biophys Acta 1823:1521-1530.).

Figure 1
Absorption and translocation of iron in rice. Adapted from Palmer and Guerinot (2009)Palmer CM and Guerinot ML (2009) Facing the challenges of Cu, Fe and Zn homeostasis in plants. Nat Chem Biol 5:333-340.; Kobayashi and Nishizawa (2012)Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.; Bashir et al. (2013a)Bashir K, Nozoye T, Ishimaru Y, Nakanishi H and Nishizawa NK (2013a) Exploiting new tools for iron bio-fortification of rice. Biotechnol Adv 31:1624-1633..

In Arabidopsis thaliana (L.) Heynh there are eight homologues of FRO (AtFRO1 to AtFRO8), while in O. sativa there are only two (OsFRO1 and OsFRO2) (Victoria et al., 2012Victoria FDC, Bervald CMP, da Maia LC, de Sousa RO, Panaud O and de Oliveira AC (2012) Phylogenetic relationships and selective pressure on gene families related to iron homeostasis in land plants. Genome 55:883-900.). The gene IRT presents 15 homologues in A. thaliana, (AtIRT1, AtIRT2, AtIRT3, AtZIP1 to AtZIP12) and 11 in O. sativa (OsIRT1, OsIRT2, OsZIP1 to OsZIP10) (Ishimaru 2005Ishimaru Y, Suzuki M, Kobayashi T, Takahashi M, Nakanishi H, Mori S and Nishizawa NK (2005) OsZIP4, a novel zinc-regulated zinc transporter in rice. J Exp Bot 56:3207-3214.; Ishimaru et al., 2006Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, et al. (2006) Rice plants take up iron as an Fe 3+ -phytosiderophore and as Fe 2+. Plant J 45:335-346.; Kim and Guerinot 2007Kim SA and Guerinot ML (2007) Mining iron: Iron uptake and transport in plants. FEBS Lett 581:2273-2280.). A. thaliana presents 12 homologues of the gene AHA (AtAHA1 to AtAHA12) (Santi and Schmidt, 2009Santi S and Schmidt W (2009) Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots. New Phytol 183:1072-1084.) and O. sativa ten (OsA1 to OsA10) (Zhu et al., 2009Zhu Y, Di T, Xu G, Chen X, Zeng H, Yan F and Shen Q (2009) Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition. Plant, Cell Environ 32:1428-1440.; Li et al., 2015Li S, Pan XX, Berry JO, Wang Y, Naren, Ma S, Tan S, Xiao W, Zhao WZ, Sheng XY, et al. (2015) OsSEC24, a functional SEC24-like protein in rice, improves tolerance to iron deficiency and high pH by enhancing H+ secretion mediated by PM-H+-ATPase. Plant Sci 233:61-71.). Not all members of FRO, ZIP and AHA families are directly involved with Fe capture (Michelet and Boutry, 1995Michelet B and Boutry M (1995) The plasma membrane H+-ATPase - A highly regulated enzyme with multiple physiological functions. Plant Physiol 108:1-6.; Bernal et al., 2012Bernal M, Casero D, Singh V, Wilson GT, Grande A, Yang H, Dodani SC, Pellegrini M, Huijser P, Connolly EL, et al. (2012) Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis. Plant Cell 24:738-761.; Milner et al., 2013Milner MJ, Seamon J, Craft E and Kochian LV (2013) Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis. J Exp Bot 64:369-381.).

In conditions of Fe deficiency there is an induction of IRT1, FRO2 and several AHAs (Colangelo and Guerinot, 2004Colangelo EP and Guerinot ML (2004) The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response. Plant Cell Online 16:3400-3412.; Santi and Schmidt, 2009Santi S and Schmidt W (2009) Dissecting iron deficiency-induced proton extrusion in Arabidopsis roots. New Phytol 183:1072-1084.; Hindt and Guerinot, 2012Hindt MN and Guerinot ML (2012) Getting a sense for signals: Regulation of the plant iron deficiency response. Biochim Biophys Acta 1823:1521-1530.). Studies conducted in A. thaliana demonstrate that the low availability of Fe leads to the induction of transcription factor (TF) FER-like iron deficiency-induced transcription factor (FIT) which regulates AtFRO2 at the level of mRNA accumulation and AtIRT1 at the level of both mRNA and protein accumulation (Eide et al., 1996Eide D, Broderius M, Fett J and Guerinot ML (1996) A novel iron-regulated metal transporter from plants identified by functional expression in yeast. Proc Natl Acad Sci USA 93:5624-5628.; Vert et al., 2002Vert G, Grotz N, Dedaldechamp F, Gaymard F, Guerinot ML, Briat JF and Curie C (2002) IRT1, an Arabidopsis transporter essential for iron uptake from the soil and plant growth. Plant Cell 14:1223-1233.; Colangelo and Guerinot, 2004Colangelo EP and Guerinot ML (2004) The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response. Plant Cell Online 16:3400-3412.). The co-expression of FIT with other TFs of the Basic helix-loop-helix (AtbHLH38/39) family directly regulates the expression of IRT1 and FRO2, increasing iron accumulation (Yuan et al., 2008Yuan Y, Wu H, Wang N, Li J, Zhao W, Du J, Wang D and Ling H-Q (2008) FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis. Cell Res 18:385-397.; Hindt and Guerinot, 2012Hindt MN and Guerinot ML (2012) Getting a sense for signals: Regulation of the plant iron deficiency response. Biochim Biophys Acta 1823:1521-1530.). There are no orthologs of FIT in rice, but AtbHLH38/39 are similar to OsIRO2 (Hindt and Guerinot 2012Hindt MN and Guerinot ML (2012) Getting a sense for signals: Regulation of the plant iron deficiency response. Biochim Biophys Acta 1823:1521-1530.) that regulates genes related to transport of Fe(III)-PS, but does not regulate OsIRT1 (Ogo et al., 2007Ogo Y, Nakanishi Itai R, Nakanishi H, Kobayashi T, Takahashi M, Mori S and Nishizawa NK (2007) The rice bHLH protein OsIRO2 is an essential regulator of the genes involved in Fe uptake under Fe-deficient conditions. Plant J 51:366-377.).

The FIT gene is regulated by signaling molecules such as auxin and ethylene, synthesized in conditions of iron deficiency. In Arabidopsis the lack of Fe induces an increase in auxin synthesis, resulting in increased expression of the genes FIT and FRO2 (Chen et al., 2010Chen WW, Yang JL, Qin C, Jin CW, Mo JH, Ye T and Zheng SJ (2010) Nitric oxide acts downstream of auxin to trigger root Ferric-Chelate Reductase activity in response to iron deficiency in Arabidopsis. Plant Physiol 154:810-819.). Similarly to what happens to auxin, an increase in ethylene synthesis is also noticed under these conditions, an event that cause the upregulation of FIT (Lucena et al., 2006Lucena C, Waters BM, Romera FJ, García MJ, Morales M, Alcántara E and Pérez-Vicente R (2006) Ethylene could influence ferric reductase, iron transporter, and H+-ATPase gene expression by affecting FER (or FER-like) gene activity. J Exp Bot 57:4145-4154.) and therefore of FRO and IRT. FIT interacts with the TFs Ethylene insensitive 3 (AtEIN3) and Ethylene insensitive 3–like1 (AtEIL1) emphasizing the importance of ethylene signaling in response to Fe deficiency (Lingam et al., 2011Lingam S, Mohrbacher J, Brumbarova T, Potuschak T, Fink-Straube C, Blondet E, Genschik P and Bauer P (2011) Interaction between the bHLH transcription factor FIT and Ethylene Insensitive3/Ethylene Insensitive3-Like1 reveals molecular linkage between the regulation of iron acquisition and ethylene signaling in Arabidopsis. Plant Cell 23:1815-1829.). It is interesting to note that there is a plethora of bHLH genes involved in iron uptake regulation and extensive additional information is available (Bashir et al., 2010Bashir K, Ishimaru Y and Nishizawa NK (2010) Iron uptake and loading into rice grains. Rice 3:122-130.; Zheng et al., 2010Zheng L, Ying Y, Wang L, Wang F, Whelan J and Shou H (2010) Identification of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa. BMC Plant Biol 10:166.; Zhao et al., 2014Zhao M, Song A, Li P, Chen S, Jiang J and Chen F (2014) A bHLH transcription factor regulates iron intake under Fe deficiency in chrysanthemum. Scientific Reports 4:6694.; Li et al., 2016Li XL, Zhang HM, Ai Q, Liang G and Yu D (2016) Two bHLH transcription factors, bHLH34 and bHLH104, regulate iron homeostasis in Arabidopsis thaliana. Plant Physiol 170:2478-2493.).

Just as auxin and ethylene, nitric oxide (NO) has its synthesis increased in conditions of Fe deficiency. NO acts as a positive regulator of genes whose products act on Fe uptake (Hindt and Guerinot, 2012Hindt MN and Guerinot ML (2012) Getting a sense for signals: Regulation of the plant iron deficiency response. Biochim Biophys Acta 1823:1521-1530.). Conversely, under conditions of Fe excess, three ZIP genes and OsFRO2 are induced in rice (Finatto et al., 2015Finatto T, de Oliveira AC, Chaparro C, da Maia LC, Farias DR, Woyann LG, Mistura CC, Soares-Bresolin AP, Llauro C, Panaud O, et al. (2015) Abiotic stress and genome dynamics: Specific genes and transposable elements response to iron excess in rice. Rice 8:13.).

In Arabidopsis the Popeye (AtPYE) and Brutus (AtBTS) genes are, respectively, a TF and an E3 ubiquitin ligase that also participate in the regulation of Fe absorption. These proteins act in sensitizing the root response to the availability of Fe, regulating Fe homeostasis (Long et al., 2010Long TA, Tsukagoshi H, Busch W, Lahner B, Salt DE and Benfey PN (2010) The bHLH transcription factor POPEYE regulates response to iron deficiency in Arabidopsis roots. Plant Cell Online 22:2219-2236.). In rice, the genes OsIRO3 (Zheng et al., 2010Zheng L, Ying Y, Wang L, Wang F, Whelan J and Shou H (2010) Identification of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa. BMC Plant Biol 10:166.) and OsHRZ1/OsHRZ2 (Kobayashi et al., 2013Kobayashi T, Nagasaka S, Senoura T, Itai RN, Nakanishi H and Nishizawa NK (2013) Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation. Nat Commun 4:1-12.), have been identified. IRO3 is an ortholog of AtPYE, and HZR1 and HZR2 are orthologs of AtBTS.

Strategy II (Figure 1) includes the participation of genes that act in the cycle of PSs precursors - METHIONINE and S-ADENOSYL-L-METHIONINE (5’-methylthioadenosine nucleosidase – MTN, Methylthioribose kinase – MTK, Methylthioribose-1-phosphate isomerase – IDI2 and dehydrase enolase phosphatase – DEP, s-adenosyl-l-methionine synthetase – SAMS) (Kobayashi et al., 2005Kobayashi T, Suzuki M, Inoue H, Itai RN, Takahashi M, Nakanishi H, Mori S and Nishizawa NK (2005) Expression of iron-acquisition-related genes in iron-deficient rice is coordinately induced by partially conserved iron-deficiency-responsive elements. J Exp Bot 56:1305-1316.; Suzuki et al., 2006Suzuki M, Takahashi M, Tsukamoto T, Watanabe S, Matsuhashi S, Yazaki J, Kishimoto N, Kikuchi S, Nakanishi H, Mori S, et al. (2006) Biosynthesis and secretion of mugineic acid family phytosiderophores in zinc-deficient barley. Plant J 48:85-97.), in the synthesis of PSs (NAS, NAAT, DMAS, Dioxygenases – IDS2/IDS3) (Nakanishi et al., 2000Nakanishi H, Yamaguchi H, Sasakuma T, Nishizawa NK and Mori S (2000) Two dioxygenase genes, Ids3 and Ids2, from Hordeum vulgare are involved in the biosynthesis of mugineic acid family phytosiderophores. Plant Mol Biol 44:199-207.; Kobayashi and Nishizawa, 2012Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.), binding of PSs to Fe(III) (Nozoye et al., 2011Nozoye T, Nagasaka S, Kobayashi T, Takahashi M, Sato Y, Sato Y, Uozumi N, Nakanishi H and Nishizawa NK (2011) Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants. J Biol Chem 286:5446-5454.), and in the transport of the complex Fe(III)-PSs into the root (YS1 and YSL) (Curie et al., 2001Curie C, Panaviene Z, Loulergue C, Dellaporta SL, Briat JF and Walker EL (2001) Maize yellow stripe1 encodes a membrane protein directly involved in Fe(III) uptake. Nature 409:346-349.; Inoue et al., 2009Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori S and Nishizawa NK (2009) Rice OsYSL15 is an iron-regulated Iron(III)-Deoxymugineic Acid Transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings. J Biol Chem 284:3470-3479.; Lee et al., 2009aLee S, Chiecko JC, Kim SA, Walker EL, Lee Y, Guerinot ML and An G (2009a) Disruption of OsYSL15 leads to iron inefficiency in rice plants. Plant Physiol 150:786-800.; Kobayashi and Nishizawa, 2012Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.). Four homologues of the gene NAS are present in arabidopsis (AtNAS1, AtNAS2, AtNAS3 and AtNAS4) and three in rice (OsNAS1, OsNAS2 and OsNAS3) (Victoria et al., 2012Victoria FDC, Bervald CMP, da Maia LC, de Sousa RO, Panaud O and de Oliveira AC (2012) Phylogenetic relationships and selective pressure on gene families related to iron homeostasis in land plants. Genome 55:883-900.). Six homologues of the NAAT gene (OsNAAT1 to OsNAAT6), and only one DMAS gene (OsDMAS1) are present in rice (Bashir et al., 2006Bashir K, Inoue H, Nagasaka S, Takahashi M, Nakanishi H, Mori S and Nishizawa NK (2006) Cloning and characterization of deoxymugineic acid synthase genes from graminaceous plants. J Biol Chem 281:32395-32402.; Widodo et al., 2010Widodo B, Broadley MR, Rose T, Frei M, Pariasca-Tanaka J, Yoshihashi T, Thomson M, Hammond JP, Aprile A, Close TJ, et al. (2010) Response to zinc deficiency of two rice lines with contrasting tolerance is determined by root growth maintenance and organic acid exudation rates , and not by zinc-transporter activity. New Phytol 400-414.). For gene YSL, eight homologues were identified in Arabidopsis (AtYSL1 to AtYSL8) and 18 in rice (OsYSL1 to YSL18) (Victoria et al., 2012Victoria FDC, Bervald CMP, da Maia LC, de Sousa RO, Panaud O and de Oliveira AC (2012) Phylogenetic relationships and selective pressure on gene families related to iron homeostasis in land plants. Genome 55:883-900.).

Like as the genes involved in strategy I, genes associated with strategy II are induced in iron deficiency (Ricachenevsky and Sperotto, 2014Ricachenevsky FK and Sperotto RA (2014) There and back again, or always there? The evolution of rice combined strategy for Fe uptake. Front Plant Sci 5:189.). The TFs Iron deficiency responsive element binding factor 1 (IDEF1 and IDEF2) and Iron regulated basic helix-loop-helix (IRO2) have been identified as regulators of key genes that control Fe uptake, including the synthesis of PSs in rice (Itai et al., 2013Itai RN, Ogo Y, Kobayashi T, Nakanishi H and Nishizawa NK (2013) Rice genes involved in phytosiderophore biosynthesis are synchronously regulated during the early stages of iron deficiency in roots. Rice 6:16.). Under Fe deficiency the OsIDEF1 upregulates genes whose products act in capture and use of Fe in rice, such as OsIRO2, OsYSL15, OsYSL2, OsIRT1, OsNAS1, OsNAS2 and OsNAS3 (Kobayashi et al., 2009Kobayashi T, Itai RN, Ogo Y, Kakei Y, Nakanishi H, Takahashi M and Nishizawa NK (2009) The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes. Plant J 60:948-961.). The TF IDEF1 binds to Iron Deficiency-responsive Element 1 (IDE1), while IDEF2 binds to IDE2, both present in the promoter region of genes associated with Fe deficiency (Kobayashi et al., 2007Kobayashi T, Ogo Y, Itai RN, Nakanishi H, Takahashi M, Mori S and Nishizawa NK (2007) The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants. Proc Natl Acad Sci U S A 104:19150-19155.; Ogo et al., 2008Ogo Y, Kobayashi T, Nakanishi Itai R, Nakanishi H, Kakei Y, Takahashi M, Toki S, Mori S and Nishizawa NK (2008) A novel NAC transcription factor, IDEF2, that recognizes the iron deficiency-responsive element 2 regulates the genes involved in iron homeostasis in plants. J Biol Chem 283:13407-13417.). Moreover, OsIRO3 is induced in Fe deficiency and acts as a negative regulator of genes related to this condition in rice (OsNAS1, OsNAS2, OsIRO2, OsIRT1, OsYSL15 and OsNRAMP1) (Zheng et al., 2010Zheng L, Ying Y, Wang L, Wang F, Whelan J and Shou H (2010) Identification of a novel iron regulated basic helix-loop-helix protein involved in Fe homeostasis in Oryza sativa. BMC Plant Biol 10:166.).

In conditions of Fe toxicity the genes OsNAS1, OsNAS2, OsYSL15, OsYSL16 and OsNRAMP1 were repressed in rice roots (Quinet et al., 2012Quinet M, Vromman D, Clippe A, Bertin P, Lequeux H, Dufey I, Lutts S and Lefèvre I (2012) Combined transcriptomic and physiological approaches reveal strong differences between short- and long-term response of rice (Oryza sativa) to iron toxicity. Plant Cell Environ 35:1837-1859.). In a similar study, Finatto et al. (2015)Finatto T, de Oliveira AC, Chaparro C, da Maia LC, Farias DR, Woyann LG, Mistura CC, Soares-Bresolin AP, Llauro C, Panaud O, et al. (2015) Abiotic stress and genome dynamics: Specific genes and transposable elements response to iron excess in rice. Rice 8:13. reported the induction of the genes OsNAAT1, OsYSL1 and OsYSL17 in rice plants grown under excessive Fe.

After Fe capture by the roots, this is transported to other organs, a process that involves several steps, passing through symplast, xylem (transpiration stream) and phloem (Kim and Guerinot, 2007Kim SA and Guerinot ML (2007) Mining iron: Iron uptake and transport in plants. FEBS Lett 581:2273-2280.). When Fe enters the symplast it is oxidized and ligated to chelating molecules (Miroslav, 1998Miroslav M (1998) The role of the redox system in uptake and translocation of iron by higher plants. Iugoslav Physiol Pharmacol Acta 34:479-489.). Chelators that can bind to Fe are, as shown in Figure 2, citrate, nicotianamine (NA) and mugineic acid (MA) (Kobayashi and Nishizawa, 2012Kobayashi T and Nishizawa NK (2012) Iron uptake, translocation, and regulation in higher plants. Annu Rev Plant Biol 63:131-152.).

Figure 2
Role of nicotianamine (NA) in iron metabolism in plant cells. Iron can enter the plant cell through various strategies depending on the nature of the iron source. In this context NA is an important chelator that is able to provide iron in a functional form, avoiding precipitation and catalysis. Adapted from Hell and Stephan (2003)Hell R and Stephan UW (2003) Iron uptake, trafficking and homeostasis in plants. Planta 216:541-551..

It has been proposed that NA facilitates Fe movement in and out of the phloem (through YSLs), while the movement of Fe within the phloem occurs via Iron Transport Proteins (ITP), dehydrins (DHN) that bind Fe3+ but not Fe2+ (Krüger et al., 2002Kruger C, Berkowitz O, Stephan UW and Hell R (2002) A metal-binding member of the late embryogenesis abundant protein family transports iron in the phloem of Ricinus communis L. J Biol Chem 277:25062-25069.; Hell and Stephan, 2003Hell R and Stephan UW (2003) Iron uptake, trafficking and homeostasis in plants. Planta 216:541-551.; Morrissey and Guerinot, 2009Morrissey J and Guerinot ML (2009) Iron uptake and transport in plants: The good, the bad, and the ionome. Chem Rev 109:4553-4567.). In A. thaliana the Ferric Reductase Defective 3 (AtFDR3) encodes a transmembrane protein belonging to the family of Multidrug and toxin efflux transporters (MATE) that facilitates the transport of citrate in the xylem (Durrett et al., 2007Durrett TP, Gassmann W and Rogers EE (2007) The FRD3-mediated efflux of citrate into the root vasculature is necessary for efficient iron translocation. Plant Physiol 144:197-205.).

In rice, a citrate transporter called OsFRDL1 is required for efficient translocation of Fe-citrate complex (Yokosho et al., 2009Yokosho K, Yamaji N, Ueno D, Mitani N and Ma JF (2009) OsFRDL1 Is a citrate transporter required for efficient translocation of iron in rice. Plant Physiol 149:297-305.). In rice plants under conditions of Fe excess, the induction of three genes belonging to the MATE family, which may be involved in reducing ROS production in mitochondria, was observed (Finatto et al., 2015Finatto T, de Oliveira AC, Chaparro C, da Maia LC, Farias DR, Woyann LG, Mistura CC, Soares-Bresolin AP, Llauro C, Panaud O, et al. (2015) Abiotic stress and genome dynamics: Specific genes and transposable elements response to iron excess in rice. Rice 8:13.). Genes belonging to YSL and IRT families, as well are not only involved in iron uptake, but also in the transport of this element through the plant. Different YSL genes transport different complexes. In rice for example, OsYSL2 transports Fe(II)NA (Koike et al., 2004Koike S, Inoue H, Mizuno D, Takahashi M, Nakanishi H, Mori S and Nishizawa NK (2004) OsYSL2 is a rice metal-nicotianamine transporter that is regulated by iron and expressed in the phloem. Plant J 39:415-424.) while OsYSL15 product transports Fe(III)-DMA (Lee et al., 2009aLee S, Chiecko JC, Kim SA, Walker EL, Lee Y, Guerinot ML and An G (2009a) Disruption of OsYSL15 leads to iron inefficiency in rice plants. Plant Physiol 150:786-800.). OsIRT1 is expressed not only in roots, but also in rice leaves and stems, indicating its participation in the Fe transport over long distances (Narayanan et al., 2007Narayanan NN, Vasconcelos MW and Grusak MA (2007) Expression profiling of Oryza sativa metal homeostasis genes in different rice cultivars using a cDNA macroarray. Plant Physiol Biochem 45:277-286.).

To be assimilated by the leaves, Fe3+ is reduced by FRO enzymes. FRO7 of A. thaliana plays a role in chloroplast iron acquisition and is required for efficient photosynthesis in young seedlings and is especially important when plants are under iron-limiting conditions (Jeong et al., 2008Jeong J, Cohu C, Kerkeb L, Pilon M, Connolly E and Guerinot ML (2008) Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions. Proc Natl Acad Sci U S A 105:10619-10624.). The LeFRO1 of Lycopersicum esculentum Mill. (Li et al., 2004Li L, Cheng X and Ling H (2004) Isolation and characterization of Fe (III)-chelate reductase gene LeFRO1 in tomato. Plant Mol Biol 54:125-136.), PsFRO1 of Pisum sativum (Waters et al., 2002Waters BM, Blevins DG and Eide DJ (2002) Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition. Plant Physiol 129:85–94.) and AtFRO6 in A. thaliana are expressed in the aerial part (Feng et al., 2006Feng H, An F, Zhang S, Ji Z, Ling H and Zuo J (2006) Light-regulated, tissue-specific, and cell differentiation-specific expression of the Arabidopsis Fe(III)-Chelate Reductase gene AtFRO6. 140:1345-1354.), indicating their participation in reduction of Fe3+. A great diversity of these proteins has been studied, and these are not only involved in iron, but also in copper homeostasis. The diverse roles of the FRO family have recently been reviewed (Jain et al., 2014Jain A, Wilson GT and Connolly EL (2014) The diverse roles of FRO family metalloreductases in iron and copper homeostasis. Front Plant Sci 5:100.).

After reduction, Fe is transported to other organs of the plant. This transport is performed via the phloem nicotianamine chelator (NA) (Takahashi et al., 2003Takahashi M, Terada Y, Nakai I, Nakanishi H, Yoshimura E, Mori S and Nishizawa NK (2003) Role of nicotianamine in the intracellular delivery of metals and plant reproductive development. Plant Cell 15:1263-1280.), which is synthesized by the enzyme Nicotianamine synthase (NAS). The Fe transport also occurs through family members of the NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN (NRAMP) (Nevo and Nelson, 2006Nevo Y and Nelson N (2006) The NRAMP family of metal-ion transporters. Biochim Biophys Acta - Mol Cell Res 1763:609-620.). NRAMP carriers are related to the subcellular transport of Fe and its partitioning in vacuoles and/or plastids (Curie et al., 2000Curie C, Alonso JM, Le Jean M, Ecker JR and Briat JF (2000) Involvement of NRAMP1 from Arabidopsis thaliana in iron transport. Biochem J 347:749-755.). Six NRAMP genes were found in Arabidopsis and eight in rice (OsNRAMP1-OsNRAMP8) (Victoria et al., 2012Victoria FDC, Bervald CMP, da Maia LC, de Sousa RO, Panaud O and de Oliveira AC (2012) Phylogenetic relationships and selective pressure on gene families related to iron homeostasis in land plants. Genome 55:883-900.).

In rice, OsNRAMP1 is expressed mainly in roots, OsNRAMP2 in leaves, and OsNRAMP3 is expressed in both tissues (Belouchi et al., 1997Belouchi A, Kwan T and Gros P (1997) Cloning and characterization of the OsNramp family from Oryza sativa, a new family of membrane proteins possibly implicated in the transport of metal ions. Plant Mol Biol 33:1085-92.). In conditions of Fe excess, Quinet et al. (2012)Quinet M, Vromman D, Clippe A, Bertin P, Lequeux H, Dufey I, Lutts S and Lefèvre I (2012) Combined transcriptomic and physiological approaches reveal strong differences between short- and long-term response of rice (Oryza sativa) to iron toxicity. Plant Cell Environ 35:1837-1859. noticed the repression of the gene OsNRAMP1, which is also involved in cadmium (Cd) accumulation (Takahashi et al., 2011aTakahashi R, Ishimaru Y, Nakanishi H and Nishizawa NK (2011a) Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice. Plant Signal Behav 6:1813-1816.,bTakahashi R, Ishimaru Y, Senoura T, Shimo H, Ishikawa S, Arao T, Nakanishi H and Nishizawa NK (2011b) The OsNRAMP1 iron transporter is involved in Cd accumulation in rice. J Exp Bot 62:4843-4850.), while Finatto et al. (2015)Finatto T, de Oliveira AC, Chaparro C, da Maia LC, Farias DR, Woyann LG, Mistura CC, Soares-Bresolin AP, Llauro C, Panaud O, et al. (2015) Abiotic stress and genome dynamics: Specific genes and transposable elements response to iron excess in rice. Rice 8:13. observed the induction of another NRAMP gene, OsNRAMP6. OsNRAMP5 is important not only for Fe, but also for manganese (Mn) and Cd transport (Ishimaru et al., 2012Ishimaru Y, Takahashi R, Bashir K, Shimo H, Senoura T, Sugimoto K, Ono K, Yano M, Ishikawa S, Arao T, et al. (2012) Characterizing the role of rice NRAMP5 in manganese, iron and cadmium transport. Sci Rep 2:286.; Sasaki et al., 2012Sasaki A, Yamaji N, Yokosho K and Ma JF (2012) Nramp5 is a major transporter responsible for manganese and cadmium uptake in rice. Plant Cell 24:2155-2167.). OsNRAMP3 is a vascular bundles-specific Mn transporter, showing once more that Mn commonly shares the same transporters with Fe in plants (Pittman, 2005Pittman JK (2005) Managing the manganese: Molecular mechanisms of manganese transport and homeostasis. New Phytol 167:733-742.; Cailliatte et al., 2010Cailliatte R, Schikora A, Briat JF, Mari S and Curie C (2010) High-affinity manganese uptake by the metal transporter NRAMP1 is essential for Arabidopsis growth in low manganese conditions. Plant Cell 22:904-917.; Yang et al., 2013Yang M, Zhang W, Dong H, Zhang Y, Lv K, Wang D and Lian X (2013) OsNRAMP3 is a vascular bundles-specific manganese transporter that is responsible for manganese distribution in rice. PLoS One 8:e83990.).

Inside the cell, Fe can be incorporated into proteins, stored in plastids and mitochondria, where it is found associated with ferritin (Duy et al., 2011Duy D, Stube R, Wanner G and Philippar K (2011) The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron. Plant Physiol 155:1709-1722.; Vigani et al., 2013Vigani G, Tarantino D and Murgia I (2013) Mitochondrial ferritin is a functional iron-storage protein in cucumber (Cucumis sativus) roots. Front Plant Sci 4:316.), or even in the vacuole of the cell (Gollhofer et al., 2014Gollhofer J, Timofeev R, Lan P, Schmidt W and Buckhout TJ (2014) Vacuolar-iron-transporter1-like proteins mediate iron homeostasis in Arabidopsis. PLoS One 9:e110468.) (Figure 1). This compartmentalization can be useful for Fe homeostasis, especially in conditions of excess of this element. Ferritin is an iron storage protein that avoids damage caused by free radicals produced by the interaction iron/dioxygen (Goto et al., 1999Goto F, Yoshihara T, Shigemoto N, Toki S and Takaiwa F (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17:282-286.). This protein has the capacity to store more than 4,500 Fe atoms in a soluble, non-toxic and bioavailable form (Briat and Lobreaux, 1997Briat JF and Lobreaux S (1997) Iron transport and storage in plants. Trends Plant Sci 2:187-193.). In Oryza glaberrima S. and O. sativa the tolerance to Fe toxicity seems to be associated with ferritin synthesis (Majerus et al., 2007Majerus V, Bertin P, Swenden V, Fortemps A, Lobréaux S and Lutts S (2007) Organ-dependent responses of the african rice to short-term iron toxicity: Ferritin regulation and antioxidative responses. Biol Plant 51:303-312.; Silveira et al., 2009Silveira VC da, Fadanelli C, Sperotto RA, Stein RJ, Basso LA, Santos DS, Vaz Junior IDS, Dias JF and Fett JP (2009) Role of ferritin in the rice tolerance to iron overload. Sci Agric 66:549-555.). A. thaliana has four homologues of ferritin encoding genes (AtFER1 to AtFER4), while in O. sativa two of these can be found (OsFER1 and OsFER2) (Silveira et al., 2009Silveira VC da, Fadanelli C, Sperotto RA, Stein RJ, Basso LA, Santos DS, Vaz Junior IDS, Dias JF and Fett JP (2009) Role of ferritin in the rice tolerance to iron overload. Sci Agric 66:549-555.). The Fe-dependent regulation of AtFER1 and ZmFER1 genes depends on the presence of a cis-element called Iron-dependent Regulatory Sequence (IDRS) in their promoter regions. The IDRS element is involved in the repression of FER genes in plants that grow under low concentrations of Fe (Petit et al., 2001Petit JM, Van Wuytswinkel O, Briat JF and Lobréaux S (2001) Characterization of an iron-dependent regulatory sequence involved in the transcriptional control of AtFer1 and ZmFer1 plant ferritin genes by iron. J Biol Chem 276:5584-5590.). In case of Fe excess, the genes OsFER1 and OsFER2 show increased amounts of transcripts, with OsFER2 being preferably upregulated (Stein et al., 2009Stein RJ, Ricachenevsky FK and Fett JP (2009) Differential regulation of the two rice ferritin genes (OsFER1 and OsFER2). Plant Sci 177:563-569.). Similar results were found by Quinet et al. (2012)Quinet M, Vromman D, Clippe A, Bertin P, Lequeux H, Dufey I, Lutts S and Lefèvre I (2012) Combined transcriptomic and physiological approaches reveal strong differences between short- and long-term response of rice (Oryza sativa) to iron toxicity. Plant Cell Environ 35:1837-1859., who also observed the induction of OsFER1 and OsFER2 genes in stress caused by excess of Fe in the soil. In other species, induction of FER genes by toxic amounts of Fe has also been observed.

Vacuoles are multifunctional organelles dynamically adjusted according to environmental conditions. This organelle has buffering capacity serving as a reservoir of metabolites, minerals, nutrients, and also as a deposit for toxic compounds, being crucial for the process of detoxification and for cellular homeostasis (Marty, 1999Marty F (1999) Plant vacuoles. Plant Cell 11:587-599.; Peng and Gong, 2014Peng J-S and Gong J-M (2014) Vacuolar sequestration capacity and long-distance metal transport in plants. Front Plant Sci 5:1-5.). The uptake of Fe by the vacuole is mediated by FERROPORTIN (FPN) (Morrissey et al., 2009Morrissey J and Guerinot ML (2009) Iron uptake and transport in plants: The good, the bad, and the ionome. Chem Rev 109:4553-4567.) and by members of a family of VACUOLAR IRON TRANSPORTERS (VIT) (Zhang et al., 2012Zhang Y, Xu YH, Yi HY and Gong JM (2012) Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. Plant J 72:400-410.). In A. thaliana three homologues of FPN (AtFPN1/AtIREG1, AtFPN2/AtIREG2 and AtFPN3/AtIREG3) were found (Curie and Briat, 2003Curie C and Briat J-F (2003) Iron transport and signaling in plants. Annu Rev Plant Biol 54:183-206.; Morrissey and Guerinot, 2009Morrissey J and Guerinot ML (2009) Iron uptake and transport in plants: The good, the bad, and the ionome. Chem Rev 109:4553-4567.; Merlot et al., 2014Merlot S, Hannibal L, Martins S, Martinelli L, Amir H, Lebrun M and Thomine S (2014) The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation. J Exp Bot 65:1551-1564.), while in O. sativa only two of these genes (OsFPN1/OsFerroportin; OsFPN2/IREG3) were detected (Bashir et al., 2011Bashir K, Ishimaru Y, Shimo H, Nagasaka S, Fujimoto M, Takanashi H, Tsutsumi N, An G, Nakanishi H and Nishizawa NK (2011) The rice mitochondrial iron transporter is essential for plant growth. Nat Commun 2:322.; Merlot et al., 2014Merlot S, Hannibal L, Martins S, Martinelli L, Amir H, Lebrun M and Thomine S (2014) The metal transporter PgIREG1 from the hyperaccumulator Psychotria gabriellae is a candidate gene for nickel tolerance and accumulation. J Exp Bot 65:1551-1564.). In Arabidopsis, iron accumulation in the vacuole of seed cells depends on AtVIT1. This protein is localized in the vacuolar membrane, and the gene is expressed in the developing embryo, seed and, in young seedlings, where the protein is predominantly associated with the vasculature (Kim et al., 2006Kim SA, Punshon T, Lanzirotti A, Li L, Alonso JM, Ecker JR, Kaplan J and Guerinot ML (2006) Localization of iron in Arabidopsis seed requires the vacuolar membrane transporter VIT1. Science 314:1295-8.). In rice the vacuolar membrane transporters encoded by OsVIT1 and OsVIT2 genes are involved in storage of iron in vacuoles of flag leaves, and the inhibition of these results in an increase of Fe in the seed, suggesting that new mechanisms are activated under this condition (Zhang et al., 2012Zhang Y, Xu YH, Yi HY and Gong JM (2012) Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. Plant J 72:400-410.), and under conditions of Fe excess, OsVIT1 was increased (Finatto et al., 2015Finatto T, de Oliveira AC, Chaparro C, da Maia LC, Farias DR, Woyann LG, Mistura CC, Soares-Bresolin AP, Llauro C, Panaud O, et al. (2015) Abiotic stress and genome dynamics: Specific genes and transposable elements response to iron excess in rice. Rice 8:13.). In Arabidopsis, Fe remobilization from the vacuole to the cytoplasm is mediated by NRAMP3 and NRAMP4 (Peng and Gong, 2014Peng J-S and Gong J-M (2014) Vacuolar sequestration capacity and long-distance metal transport in plants. Front Plant Sci 5:1-5.).

Quantitative Trait Loci

In anaerobic conditions, high amounts of Fe2+ are taken up by plants, resulting in the accumulation of this element in the cell (Santos and de Oliveira, 2007Santos LS and de Oliveira AC (2007) Rice iron metabolism: From source to solution. J Crop Sci Biotechnol 10:64-72.). In rice, there is a differential response among cultivars to stress by Fe excess. When both susceptible and tolerant cultivars, BR-IRGA 409 and EPAGRI 108 respectively, were subjected to high concentrations of Fe there was less accumulation of this element and greater accumulation of ferritin in the tolerant cultivar, suggesting that this protein may be involved in this mechanism of tolerance (Silveira et al., 2009Silveira VC da, Fadanelli C, Sperotto RA, Stein RJ, Basso LA, Santos DS, Vaz Junior IDS, Dias JF and Fett JP (2009) Role of ferritin in the rice tolerance to iron overload. Sci Agric 66:549-555.). However, a study by Panda et al. (2014)Panda BB, Sharma S, Mohapatra PK and Das A (2014) Iron nutrition vis-à-vis aconitase activity and ferritin accumulation in tropical Indica Rice cultivars differing in grain iron concentration. Am J Plant Sci 5:2829-2841. found that when there is Fe accumulation, the activity of aconitase and ferritin levels are higher in a cultivar that accumulates higher concentrations of Fe compared to the cultivar that has a lower concentration of this element. It is also interesting to highlight that a previous study showed that the accumulation of iron is not parallel to the level of ferritin expression in rice seeds overexpressing the SoyFER gene (of soybean ferritin), suggesting that Fe accumulation may be limited by the uptake and transport of this element (Qu et al., 2005Qu LQ, Yoshihara T, Ooyama A, Goto F and Takaiwa F (2005) Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds. Planta 222:225-33.). According to these studies, the mechanisms associated with tolerance to toxicity and accumulation of Fe are not well understood. However, studies related to the identification of Quantitative Trait Loci (QTLs) and genes whose products are responsible for the homeostasis of Fe and the accumulation of this mineral in the grain have been conducted (Figure 3 and Table S1), and the results of these surveys can assist breeding programs for toxicity tolerance, as well as biofortification for Fe content (Wu et al., 1998Wu P, Hu B, Liao CY, Zhu JM, Wu YR, Senadhira D and Paterson AH (1998) Characterization of tissue tolerance to iron by molecular markers in different lines of rice. Plant and Soil 203:217-226.; Wan et al., 2003Wan JL, Zhai HQ and Ikehashi H (2003) Detection and analysis of QTLs for ferrous iron toxicity tolerance in rice, Oryza sativa L. Euphytica 131:201-206.; Dufey et al., 2009Dufey I, Hakizimana P, Draye X, Lutts S and Bertin P (2009) QTL mapping for biomass and physiological parameters linked to resistance mechanisms to ferrous iron toxicity in rice. Euphytica 167:143-160.; Shimizu, 2009Shimizu A (2009) QTL analysis of genetic tolerance to iron toxicity in rice (Oryza sativa L.) by quantification of bronzing score. J New Seeds 10:171-179.; Wu et al., 2014Wu L, Shhadi MY, Gregorio G, Matthus E, Becker M and Frei M (2014) Genetic and physiological analysis of tolerance to acute iron toxicity in rice. Rice 7:8.). In this regard, three loci were identified on rice chromosomes 7, 8 and 9 that explain around 19-30% of the difference in the concentration of Fe in grains (Gregorio et al., 2000Gregorio G, Senadhira D, Htut H and Graham RR (2000) Breeding for trace mineral density in rice. Food Nutr Bull 21:382-386.). Another study that did not analyze QTLs but gene expression, showed that higher concentrations of Fe in grains were positively correlated with the expression of the genes OsYSL14, OsNAC5, and negatively correlated with OsNRAMP7, OsNRAMP8 and OsFRO1 expression (Sperotto et al., 2010Sperotto RA, Boff T, Duarte GL, Santos LS, Grusak MA and Fett JP (2010) Identification of putative target genes to manipulate Fe and Zn concentrations in rice grains. J Plant Physiol 167:1500-1506.). On the other hand, OsFER1, OsNRAMP4, OsNRAMP5, OsNRAMP6, OsYSL6, OsYSL12, OsYSL4, OsZIP8, OsZIP10 were correlated with higher concentration of Fe in grains. The functional characterization of these genes can help in getting biofortified rice genotypes with higher concentrations of Fe in grains. In a QTL analysis for tolerance to bronzing, using an F3 population from the cross between cv. Gimbozu (japonica genotype which is tolerant to Fe excess) and cv. Kasalath (indica genotype which is susceptible to Fe excess), seven QTLs associated with this feature were detected. These QTLs, which are located on chromosomes 1, 2, 7, 8 and 12, explain 99% of the phenotypic variation for bronzing and showed no detectable epistatic effect (Shimizu, 2009Shimizu A (2009) QTL analysis of genetic tolerance to iron toxicity in rice (Oryza sativa L.) by quantification of bronzing score. J New Seeds 10:171-179.). In a population generated from the cross between cv. Azucena (tolerant japonica) and cv. IR64 (susceptible indica), a QTL on chromosome 1 was found associated with leaf bronzing index (Dufey et al., 2009Dufey I, Hakizimana P, Draye X, Lutts S and Bertin P (2009) QTL mapping for biomass and physiological parameters linked to resistance mechanisms to ferrous iron toxicity in rice. Euphytica 167:143-160.). The association of this region with the bronzing index had already been detected earlier (Wan et al., 2003Wan JL, Zhai HQ and Ikehashi H (2003) Detection and analysis of QTLs for ferrous iron toxicity tolerance in rice, Oryza sativa L. Euphytica 131:201-206.; Wu et al., 1998Wu P, Hu B, Liao CY, Zhu JM, Wu YR, Senadhira D and Paterson AH (1998) Characterization of tissue tolerance to iron by molecular markers in different lines of rice. Plant and Soil 203:217-226.). Also in a QTL analysis in a population obtained from the cross between cv. Kasalath (susceptible indica) and cv. Koshihikari (tolerant japonica), a QTL on chromosome 3 was found associated with Fe concentration in the shoot (Fukuda et al., 2012Fukuda A, Shiratsuchi H, Fukushima A, Yamaguchi H, Mochida H, Terao T and Ogiwara H (2012) Detection of chromosomal regions affecting iron concentration in rice shoots subjected to excess ferrous iron using chromosomal segment substitution lines between Japonica and Indica. Plant Prod Sci 15:183-191.). In a study conducted by Wu et al. (2014)Wu L, Shhadi MY, Gregorio G, Matthus E, Becker M and Frei M (2014) Genetic and physiological analysis of tolerance to acute iron toxicity in rice. Rice 7:8., populations from the crosses IR29 (susceptible indica) x Pokkali (tolerant indica) and Nipponbare (moderately tolerant japonica) x Kasalath (highly susceptible japonica) were used for identification of QTLs associated with tolerance to Fe excess. In the population IR29/Pokkali the authors identified seven QTLs for leaf bronzing, located on chromosomes 1, 2, 4, 7 and 12, explaining 9.2 to 18.7% of the phenotypic variation. In a Nipponbare/Kasalath/Nipponbare backcross inbred population, three QTLs were mapped on chromosomes 1, 3 and 8, and these QTLs explain 11.6 to 18.6% of the phenotypic variation. Additional studies demonstrated that the QTL on chromosome 1 was associated with shoot tolerance, and the QTL on chromosome 3 was associated with exclusion of Fe in roots. Similarly to the QTL studies for stress tolerance to Fe, much effort has been made in identifying QTLs associated with Fe content in grains. Four QTLs for Fe accumulation (qFe1, qFe3, qFe4 and qFe7) located on chromosomes 1, 3, 4 and 7, accounting, respectively, for 16.2%, 21.4%, 9.7% and 15.5% of the phenotypic variation, were found in an F6 population from the cross cv. Bala (indica) x Azucena (japonica) (Norton et al., 2010Norton GJ, Deacon CM, Xiong L, Huang S, Meharg AA and Price AH (2010) Genetic mapping of the rice ionome in leaves and grain: Identification of QTLs for 17 elements including arsenic, cadmium, iron and selenium. Plant Soil 329:139-153.). Using Composite Interval Mapping on an F6 population from the cross Madhukar x Swarna, it was possible to identify seven QTLs associated with iron accumulation (qFe1.1, qFe1.2, qFe5.1, qFe7.1, qFe7.2, qFe12.1 e qFe12.2), which are located on chromosomes 1, 5, 7 and 12 (Anuradha et al., 2012Anuradha K, Agarwal S, Rao YV, Rao KV, Viraktamath BC and Sarla N (2012) Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar Swarna RILs. Gene 508:233-240.). The candidate genes in these QTLs are: OsYSL1 (LOC_Os01g13710), which is located within qFe1.2; OsMTP1 (LOC_Os05g03780) located within qFe5.1; OsNas3 (LOC_Os07g48980) located within qFe7.1 and qFe7.2; OsNRAMP1 (LOC_Os07g15460) located within qFe7.2; and OsZIP8 (LOC_Os07g12890) located 0.3 Mb right of qFe12.1. Most phenotypic variance was explained by the QTL on chromosome 12 (71%) (Anuradha et al., 2012Anuradha K, Agarwal S, Rao YV, Rao KV, Viraktamath BC and Sarla N (2012) Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar Swarna RILs. Gene 508:233-240.).

Figure 3
QTLs related to Fe metabolism. Map with the location of different QTLs related to tolerance to low and/or excessive amounts of Fe in the soil, and/or related to the variation of Fe content in grains.

Mapping of a population derived from the cross Chunjiang 06 (japonica) x TN1 (indica) detected three QTLs for Fe accumulation in grains. The QTLs are located on chromosomes 1, 6 and 8, explaining, respectively 15.7, 10.6 and 22.3% of the phenotypic variation for Fe accumulation in grains (Du et al., 2013Du J, Zeng D, Wang B, Qian Q, Zheng S and Ling HQ (2013) Environmental effects on mineral accumulation in rice grains and identification of ecological specific QTLs. Environ Geochem Health 35:161-170.). A QTL related to Fe concentration, was detected on chromosome 8 through the study of a population from the cross cv. Lemont (japonica) x cv. TeQing (indica) (Zhang et al., 2014Zhang M, Pinson SRM, Tarpley L, Huang XY, Lahner B, Yakubova E, Baxter I, Guerinot ML and Salt DE (2014) Mapping and validation of quantitative trait loci associated with concentrations of 16 elements in unmilled rice grain. Theor Appl Genet 127:137-165.). A collection of Dale Bumpers National Rice Research Center of the USDA ARS, Stuttgart, AR, USA composed by 221 accesses of O. sativa, five accesses of O. glaberrima, two accesses of Oryza rufipogon Griff. and one of Oryza nivara Sharma et Shastry has been mapped aiming the identification of QTLs for different contents of minerals in the grain (Nawaz et al., 2015Nawaz Z, Kakar KU, Li X, Li S, Zhang B, Shou H and Shu Q (2015) Genome-wide association mapping of Quantitative Trait Loci (QTLs) for contents of eight elements in Brown Rice (Oryza sativa L.). J Agric Food Chem 63:8008-8016.). In this study, the authors identified 11 genetic regions responsible for binding and transport of Fe, comprising the genes OsZIP1 (Os01g0972200), OsHMA4 (Os02g0196600), OsACA2 (Os02g0176700), OsZIP2 (Os03g0411800), OsCNGC (Os03g0758300), OsZIP3 (Os04g0613000), OsZIP5 (Os05g0472700), OsZIP9 (Os05g0472400), OsHma2 (Os06g0700700), Abc transporter (Os06g0607700), OsNAS3 (Os07g0677300), Heavy metal transporter (Os07g0671400), Chy zinc finger (Os10g0456800) and OsACA9 (Os12g0136900). In A. thaliana, two QTLs were identified on chromosomes 1 and 5, in a region in which genes (ZIP10 and NAS1) are associated with Fe, playing a role in cation translocation (Vreugdenhil et al., 2004Vreugdenhil D, Aarts MGM, Koornneef M, Nelissen H and Ernst WHO (2004) Natural variation and QTL analysis for cationic mineral content in seeds of Arabidopsis thaliana. Plant Cell Environ 27:828-839.). Although further studies are required for the elucidation of mechanisms and genes related with the increase of iron concentration in seeds and stress tolerance for Fe excess, much work has already been developed in QTL mapping and its association with other metabolic pathways (Wan et al., 2003Wan JL, Zhai HQ and Ikehashi H (2003) Detection and analysis of QTLs for ferrous iron toxicity tolerance in rice, Oryza sativa L. Euphytica 131:201-206.; Shimizu, 2009Shimizu A (2009) QTL analysis of genetic tolerance to iron toxicity in rice (Oryza sativa L.) by quantification of bronzing score. J New Seeds 10:171-179.).

Phylogeny

A phylogenetic study on members of gene families related to Fe homeostasis (NAS, NRAMP, YSL, FRO and IRT) was conducted in O. sativa, A. thaliana, Physcomitrella patens (Hedw.) Bruch & Schimp. and other monocots and dicots (Victoria et al., 2012Victoria FDC, Bervald CMP, da Maia LC, de Sousa RO, Panaud O and de Oliveira AC (2012) Phylogenetic relationships and selective pressure on gene families related to iron homeostasis in land plants. Genome 55:883-900.). In this study, the authors found that FRO genes can be grouped into two clusters, but these do not separate monocots, dicots and bryophytes, a first clue indicating that the divergence of these genes occurred even before the diversification of land plants. Conversely, for NAS genes the formation of a group with monocots and dicots was observed. In the IRT family the genes were grouped into different clusters that separate monocots, dicots and bryophytes. For NRAMP genes, no evidence for divergence between groups of plants was observed, since genes from monocots and dicots were together in different clusters. Finally, the authors found that YSL genes possibly went through two duplication events, which probably occurred before the divergence of monocots and dicots (Victoria et al., 2012Victoria FDC, Bervald CMP, da Maia LC, de Sousa RO, Panaud O and de Oliveira AC (2012) Phylogenetic relationships and selective pressure on gene families related to iron homeostasis in land plants. Genome 55:883-900.).

Phylogenetic analyses were also performed by Gross et al. (2003)Gross J, Stein RJ, Fett-Neto AG and Fett JP (2003) Iron homeostasis related genes in rice. Genet Mol Biol 26:477-497.. In this study they analyzed a total of 43 genes belonging to five families: YS, FRO, ZIP, NRAMP, and ferritin proteins. The analysis of the YS family showed a relationship between predicted members of rice, Arabidopsis and maize (Zea mays L.), indicating that the putative new genes were homologous to maize YS, indicating that these may also have a role in Fe transport. The proteins from family FRO were separated from the burst oxidases, with a subdivision of FRO sequences, having OsFRO1 in one group and OsFRO2 in another. Members of the ZIP family were grouped in a single tree, with OsZIP1 and OsZIP6 more distantly related. The NRAMP family was divided into two classes, one more similar to AtNRAMP1 and another to AtNRAMP2, in which the number of exons is determinative in grouping these sequences. The ferritin family showed a separation between each of the species analyzed, where mammalian ferritins were separated from their respective homologues. The separation was also noticed between monocots and dicots, and first we can observe the divergence of Arabidopsis genes, before genes from maize and rice diverge from each other.

Strategies for Fe biofortification in rice

Biofortification is a process that increases the bioavailability of essential elements in the edible part of plants (White and Broadley, 2005White P and Broadley M (2005) Biofortifying crops with essential mineral elements. Trends Plant Sci 10:586-593.; Zielinska-Dawidziak, 2015Zielinska-Dawidziak M (2015) Plant ferritin - A source of iron to prevent its deficiency. Nutrients 7:1184-1201.). Although Fe is the fourth most abundant element in the earth’s crust, little of this element is available for human nutrition by grains (Kim and Guerinot, 2007Kim SA and Guerinot ML (2007) Mining iron: Iron uptake and transport in plants. FEBS Lett 581:2273-2280.), a fact that contributes to ranking iron deficiency as the sixth risk factor for death and disability (WHO, 2015WHO (2015) World Health Organization, http://www.who.int/nutrition/publications/en/ida_assessment_prevention_control.pdf (December 05, 2015).
http://www.who.int/nutrition/publication...
).

Although rice is a widely consumed food, it is not a rich source of iron, furthermore most of the Fe content of rice grains is accumulated in the aleurone and in the embryo, two parts that are lost during milling. After that, grains consist almost in its entirety of the endosperm, having lost up to 80% of the iron content and constituting a poor source of Fe for the human diet. This makes the evaluation of iron content in polished and unpolished grains an important piece of information when studying biofortification (Brinch-Pedersen et al., 2007Brinch-Pedersen H, Borg S, Tauris B and Holm PB (2007) Molecular genetic approaches to increasing mineral availability and vitamin content of cereals. J Cereal Sci 46:308-326.; Paul et al., 2012Paul S, Ali N, Gayen D, Datta SK and Datta K (2012) Molecular breeding of Osfer 2 gene to increase iron nutrition in rice grain. GM Crops Food 3:310-6.; Bashir et al., 2013bBashir K, Takahashi R, Akhtar S, Ishimaru Y, Nakanishi H and Nishizawa N (2013b) The knockdown of OsVIT2 and MIT affects iron localization in rice seed. Rice 6:31-37.).

Among plant breeding methods, transgenesis has high potential for Fe biofortification since this is a fast and efficient technique that is already being used for this purpose. Studies on rice biofortification by Fe using transgenesis were conducted using five different strategies. In the first strategy, the increase in the amount of Fe in the grain was achieved through the expression of soybean ferritin (SoyFerh1) under control of the Glutelin gene promoter from rice (OsGLUB1), which is specific for the endosperm. The higher expression of ferritin in the endosperm resulted in an at least two-fold increase of Fe in japonica cv. Kitaake (Goto et al., 1999Goto F, Yoshihara T, Shigemoto N, Toki S and Takaiwa F (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17:282-286.) and in japonica cv. Taipei 309 (Lucca et al., 2001Lucca P, Hurrell R and Potrykus I (2001) Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains. Theor Appl Genet 102:392-397.). The increase was 3.7-fold in indica cv. IR68144 (Vasconcelos et al., 2003Vasconcelos M, Datta K, Oliva N, Khalekuzzaman M, Torrizo L, Krishnan S, Oliveira M, Goto F and Datta SK (2003) Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene. Plant Sci 164:371-378.) and 2.1-fold in indica cv. Pusa Sugandhi II (Paul et al., 2012Paul S, Ali N, Gayen D, Datta SK and Datta K (2012) Molecular breeding of Osfer 2 gene to increase iron nutrition in rice grain. GM Crops Food 3:310-6.).

In the second strategy, the increase in the amount of Fe in grains was due to the overexpression of genes involved in the synthesis of mugineic acid. When overexpressing Nicotianamine synthase (NAS) it was possible to notice an Fe content increase of even more than threefold in polished grains of the japonica cultivars Tsukinohikari (Masuda et al., 2009Masuda H, Usuda K, Kobayashi T, Ishimaru Y, Kakei Y, Takahashi M, Higuchi K, Nakanishi H, Mori S and Nishizawa NK (2009) Overexpression of the barley nicotianamine synthase gene HvNAS1 increases iron and zinc concentrations in rice grains. Rice 2:155-166.), Dongjin (Lee et al., 2009bLee S, Jeon US, Lee SJ, Kim YK, Persson DP, Husted S, Schjørring JK, Kakei Y, Masuda H, Nishizawa NK, et al. (2009b) Iron fortification of rice seeds through activation of the nicotianamine synthase gene. Proc Natl Acad Sci U S A 106:22014-22019.), and Nipponbare (Johnson et al., 2011Johnson AAT, Kyriacou B, Callahan DL, Carruthers L, Stangoulis J, Lombi E and Tester M (2011) Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS One 6:e24476.). When Dioxigenase (IDS3) was overexpressed it caused an Fe content increase of 1.4-fold in polished grains of the japonica rice Tsukinohikari (Masuda et al., 2008Masuda H, Suzuki M, Morikawa KC, Kobayashi T, Nakanishi H, Takahashi M, Saigusa M, Mori S and Nishizawa NK (2008) Increase in Iron and zinc concentrations in rice grains via the introduction of barley genes involved in phytosiderophore synthesis. Rice 1:100-108.).

In the third strategy, the OsYSL2 gene was inserted under the control of the promoter of Sucrose transporter (OsSUT1), resulting in increased expression of this gene in panicle and grains. This transformation increased by 4.4-fold the concentration of Fe in polished grains of the japonica cultivar Tsukinohikari (Ishimaru et al., 2010Ishimaru Y, Masuda H, Bashir K, Inoue H, Tsukamoto T, Takahashi M, Nakanishi H, Aoki N, Hirose T, Ohsugi R, et al. (2010) Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese. Plant J 62:379-390.). The fourth strategy is a combination of the first three, generating the rice “FER-NAS-YSL2”, which presented a 4 to 6-fold increase in Fe content in polished grains of japonica cv. Tsukinohikari (Masuda et al., 2012Masuda H, Ishimaru Y, Aung MS, Kobayashi T, Kakei Y, Takahashi M, Higuchi K, Nakanishi H and Nishizawa NK (2012) Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition. Sci Rep 2:1-7.), and a 3.4-fold increase in the other japonica cv. Paw Yin San (Aung et al., 2013Aung MS, Masuda H, Kobayashi T, Nakanishi H, Yamakawa T and Nishizawa NK (2013) Iron biofortification of myanmar rice. Front Plant Sci 4:158.).

Here it is interesting to note that Johnson et al. (2011)Johnson AAT, Kyriacou B, Callahan DL, Carruthers L, Stangoulis J, Lombi E and Tester M (2011) Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS One 6:e24476. generated three populations of rice constitutively overexpressing OsNAS1, OsNAS2 or OsNAS3. In this study nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grains of all of these three overexpression populations, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations.

Trijatmiko et al. (2016)Trijatmiko KR, Dueñas C, Tsakirpaloglou N, Torrizo L, Arines FM, Adeva C, Balindong J, Oliva N, Sapasap MV, Borrero J, et al. (2016) Biofortified indica rice attains iron and zinc nutrition dietary targets in the field. Sci Rep 6:19792. evaluated polished grains of transgenic events grown in field conditions in two countries and showed that event NASFer-274 (containing OsNAS2 and soybean ferritin (SferH-1) genes) showed good results without yield penalty or altered grain quality.

In the fifth strategy, besides increasing the Fe content in the grain, it was sought to increase tolerance to Fe deficiency as well. In this case, a concurrent insertion was used, with the SoyFERH2 gene under the control of promoters of OsGLUB1 and OsGLB, and also the HvNAS1 genes Nicotianamine aminotransferase (HvNAAT-A and HvNAAT-B) and Mugineic acid synthase (IDS3) of barley, which encode enzymes for the biosynthesis of MAs. Here the transformed plants were tolerant to Fe deficiency and also capable of accumulating 2.5 to 4-fold of this mineral in polished grains (Masuda et al., 2013Masuda H, Kobayashi T, Ishimaru Y, Takahashi M, Aung MS, Nakanishi H, Mori S and Nishizawa NK (2013) Iron-biofortification in rice by the introduction of three barley genes participated in mugineic acid biosynthesis with soybean ferritin gene. Front Plant Sci 4:132.).

Also, the overexpression of the gene OsIRT1 using a constitutive promoter (maize ubiquitin), resulted in higher concentration of iron and zinc in shoots and roots and an increase in tolerance to iron deficiency at the seedling stage. It was also possible to detect an increase in the concentration of these metals in mature grains, with 13% more iron and 12% more zinc (Lee and An, 2009Lee S and An G (2009) Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice. Plant Cell Environ 32:408-416.).

Similar data were found in plants overexpressing OsIRO2. These plants were shown to be more tolerant to iron deficiency and presented an increase in Fe content in shoots (two-fold increase) and grains (more than twice) when grown in calcareous soil (Ogo et al., 2011Ogo Y, Itai RN, Kobayashi T, Aung MS, Nakanishi H and Nishizawa NK (2011) OsIRO2 is responsible for iron utilization in rice and improves growth and yield in calcareous soil. Plant Mol Biol 75:593-605.).

In addition, another strategy used is the knockdown of the gene OsVIT2, an important gene in the increase of iron concentration (Zhang et al., 2012Zhang Y, Xu YH, Yi HY and Gong JM (2012) Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. Plant J 72:400-410.). Bashir et al. (2013b)Bashir K, Takahashi R, Akhtar S, Ishimaru Y, Nakanishi H and Nishizawa N (2013b) The knockdown of OsVIT2 and MIT affects iron localization in rice seed. Rice 6:31-37. showed that transgenic OsVIT2-knockdown plants had an increase (1.8-fold) in the concentration of iron in polished grains. This suggests that the disruption of this gene helps in increasing the amount of iron in the grains, constituting a possible strategy for producing biofortified rice.

Although the strategies using transgenesis resulted in an increase in Fe content in grains, it is known that the polishing process is still responsible for major losses of this mineral. However, we should not forget that, the location of Fe in the grain may vary according to genotype (Doesthale et al., 1979Doesthale Y, Devara S, Rao S and Belavady B (1979) Effect of milling on mineral and trace element composition of raw and parboiled rice, J Sci Food Agric 30:40-46.; Sperotto et al., 2010Sperotto RA, Boff T, Duarte GL, Santos LS, Grusak MA and Fett JP (2010) Identification of putative target genes to manipulate Fe and Zn concentrations in rice grains. J Plant Physiol 167:1500-1506.). Thus, further studies should be conducted aiming to develop new strategies for internalization of Fe (Sperotto, 2013Sperotto RA (2013) Zn/Fe remobilization from vegetative tissues to rice seeds: Should I stay or should I go? Ask Zn/Fe supply! Front Plant Sci 4:464.).

The flag leaves are the main source of photoassimilates for the development of seeds in rice. The Fe concentration of the flag leaf decreases during the reproductive development in rice, whereas the iron content of the grains increases. An interesting fact is that cultivars with lower Fe accumulation in grains show higher Fe accumulation in flag leaves. This was demonstrated in a study that showed that there is an iron remobilization from the flag leaves to the grains, and increasing this remobilization can help in obtaining biofortified grains (Sperotto et al., 2010Sperotto RA, Boff T, Duarte GL, Santos LS, Grusak MA and Fett JP (2010) Identification of putative target genes to manipulate Fe and Zn concentrations in rice grains. J Plant Physiol 167:1500-1506.). Still it is interesting to remember that other studies conducted by the same group showed that flag leaf removal (at anthesis) under field conditions did not affect seed Fe and Zn accumulation, suggesting that the flag leaves can be important, but not necessary, unless under low iron supply from the soil (Sperotto et al., 2013Sperotto RA, Ricachenevsky FK, Waldow VA, Müller ALH, Dressler VL and Fett JP (2013) Rice grain Fe, Mn and Zn accumulation: How important are flag leaves and seed number? Plant Soil Environ 59:262-266.; Sperotto, 2013Sperotto RA (2013) Zn/Fe remobilization from vegetative tissues to rice seeds: Should I stay or should I go? Ask Zn/Fe supply! Front Plant Sci 4:464.).

The commercialization of genetically modified Fe biofortified crops has some limitations, either by farmers (changes in the appearance of the product) and consumers (high cost and acceptance of genetically modified organisms). In this sense, methods based on the selection of genotypes that are rich in Fe, followed by hybridization, may be better accepted (Zielinska-Dawidziak, 2015Zielinska-Dawidziak M (2015) Plant ferritin - A source of iron to prevent its deficiency. Nutrients 7:1184-1201.).

Rice Germplasm banks can be screened to identify genotypes that can absorb and store Fe more efficiently, so more QTLs related to these characteristics can be mapped and introgressed in elite varieties. In this case, one needs to take into account the natural variation that occurred during evolution, taking advantage of the effects of specific interactions between different genes and alleles (Schuler and Bauer, 2012Schuler M and Bauer P (2012) Strategies for iron biofortification of crop plants. In: Kapiris K (ed) Food Quality, InTech. Available from: http://www.intechopen.com/books/food-quality/strategies-for-iron-biofortification-of-crop-plants.
http://www.intechopen.com/books/food-qua...
; Pinson et al., 2015Pinson SRM, Tarpley L, Yan W, Yeater K, Lahner B, Yakubova E, Huang X, Zhang M, Guerinot ML and Salt DE (2015) Worldwide genetic diversity for mineral element concentrations in rice grain. Crop Sci 55:294-311.). An example of the potential for exploitation of these banks is the 4-fold difference found when comparing the iron content of aromatic and traditional varieties (Mulualem, 2015Mulualem T (2015) Application of bio-fortification through plant breeding to improve the value of staple crops. Biomedicine and Biotechnology 3:11-19.).

The natural variation related to Fe accumulation in rice grains that was already detected is quite low. In addition, grinding and polishing the grains results in a loss of up to 80% of this element (Brinch-Pedersen et al., 2007Brinch-Pedersen H, Borg S, Tauris B and Holm PB (2007) Molecular genetic approaches to increasing mineral availability and vitamin content of cereals. J Cereal Sci 46:308-326.). Furthermore, the Fe concentration is deeply influenced by the interaction between genotype and environment (Graham et al., 1999Graham R, Senadhira D, Beebe S, Iglesias C and Monasterio I (1999) Breeding for micronutrient density in edible portions of staple food crops: Conventional approaches. F Crop Res 60:57-80.). However, despite these limitations, the International Rice Research Institute (IRRI) has developed the cultivar IR68144, which has about twice the concentration of Fe when compared with local varieties used in the Philippines (Gregorio et al., 2000Gregorio G, Senadhira D, Htut H and Graham RR (2000) Breeding for trace mineral density in rice. Food Nutr Bull 21:382-386.).

The development of cultivars with increased iron content in the grains, even at relatively low levels, associated with results of the characterization of 1,138 genotypes, that identified a variation of 6.3 to 24.4 μg.g-1 of Fe in grains, suggests that there is genetic potential for the development of other, new varieties with high accumulation of Fe (Gregorio et al., 2000Gregorio G, Senadhira D, Htut H and Graham RR (2000) Breeding for trace mineral density in rice. Food Nutr Bull 21:382-386.; Mulualem, 2015Mulualem T (2015) Application of bio-fortification through plant breeding to improve the value of staple crops. Biomedicine and Biotechnology 3:11-19.). Furthermore, the genetic variability for the content of phytic acid can also be exploited, and these possibilities make the future of genetic progress seem really optimistic (Liu, 2005Liu Z (2005) Grain phytic acid content in japonica rice as affected by cultivar and environment and its relation to protein content. Food Chem 89:49-52.).

Conclusions

Being essential in the composition of different proteins and metabolic pathways, iron is vital for animal and plant health. Actually, it is an element capable of generating toxic effects due to its high bioavailability and is also a problem due to its low availability. To solve this problem, studies aiming the identification and understanding of pathways related to the regulation of iron metabolism are being conducted, combined with molecular markers in the identification of QTLs associated with these pathways. Furthermore, phylogeny can be used to better understand the evolution of the involved genes aiming not only to decrease the sensitivity of rice both to the lack and to the excess of iron in the soil, but also to help in the generation of biofortified plants with higher iron content in the grains.

Looking at these studies it is possible to see success, not only in the description of regulatory pathways, but also in breeding for improved varieties. Advances continue to be made and obstacles being overcome. In the future we should add efforts towards identifying more QTLs related to iron excess tolerance, and to increase iron content in grains. This, allied to the exploration of the existing variation for genes that have proven to be important in experiments involving transgenic analysis, should enable us to achieve greater market acceptance and to reduce bureaucratic obstacles, which greatly hinder the release of genetically modified organisms.

Although the genetic progress may seem difficult at certain times, our ability to deal with iron metabolism in rice has increased, and soon we should obtain cultivars that will be highly tolerant to iron stress, both against excess and lack of this mineral, and, allied to this, we should also be able to develop biofortified plants with higher content of iron in their grains, helping in the fight against anemia and providing better quality of life to humanity.

Acknowledgments

This work was supported by the Brazilian Ministry of Science and Technology, National Counsel of Technological and Scientific Development (CNPq); Coordination for the Improvement of Higher Education Personnel (CAPES) and the Rio Grande do Sul State Foundation for Research Support (FAPERGS).

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Internet Resources

  • Associate Editor: Marcia Pinheiro Margis

Supplementary Material

The following online material is available for this article:

Table S1 - Positions of the QTLs related to Fe metabolism shown in Figure 3.

Publication Dates

  • Publication in this collection
    16 Mar 2017
  • Date of issue
    2017

History

  • Received
    18 Feb 2016
  • Accepted
    22 Sept 2016
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