Establishment of a non-transgenic iron-biofortified rice line using a novel HRZ1 mutation

Establishment of a non-transgenic iron-biofortified rice line using a novel HRZ1 mutation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment of a non-transgenic iron-biofortified rice line using a novel HRZ1 mutation Akihiro Saito, Junya Kumano, Masataka Suzuki, Kento Nakamura, and 22 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7955540/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2026 Read the published version in Rice → Version 1 posted 11 You are reading this latest preprint version Abstract Iron (Fe) deficiency anemia is a significant public health problem worldwide. The development of Fe biofortification in staple food crops, such as rice, through non-transgenic methods is highly anticipated to enhance broad applicability. In this study, we isolated a high Fe-accumulating mutant ( tetsu ) from an N -methyl- N -nitrosourea (MNU) mutagenized ‘Taichung-65 (T65)’ rice population. The tetsu mutant accumulated more than 3-fold higher levels of Fe and significantly higher levels of manganese (Mn) and nickel (Ni) in the shoot than the wild-type T65, whereas the levels of toxic heavy metals such as cadmium (Cd), lead (Pb), and cobalt (Co) were comparable to those of the wild-type. In both polished and brown rice of the tetsu mutant, Fe increased by approximately 2-fold, and Zn and Cu also significantly increased compared with those in T65. Perls’ staining revealed that Fe localization in rice grains was not limited to the outer layers and scutellum, but also extended into the endosperm of the tetsu mutant. Concomitant with high Fe accumulation, the tetsu mutant showed remarkable tolerance to alkaline Fe-deficient soil conditions. Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) analysis revealed a novel nonsense mutation in the Hemerythrin motif-containing Ring Zinc-finger protein 1 ( HRZ1 ) gene in the tetsu genome, which is known to govern the negative regulation of the Fe deficiency response and is crucial for normal development. The homozygous tetsu mutation leads to a substantial increase in shoot Fe content, alongside the upregulation of several genes related to Fe uptake and translocation, without causing serious adverse effects on growth. To utilize this novel mutation in Fe-biofortified rice breeding, we created recombinant inbred lines (RILs) derived from crosses between the tetsu mutant and ‘Asamurasaki,’ a nutrient-rich black rice cultivar. During the breeding process, we successfully selected RILs that exhibited normal growth and fertility, resulting in the development of non-transgenic Fe-biofortified rice lines with various waxy/glutinous properties and polyphenol content in brown rice for versatile applications. These results indicate that the identified novel HRZ1 mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars with various beneficial traits. Fe-biofortified plant Fe uptake and accumulation Fe-deficiency anemia HRZ1 Rice (Oryza sativa) GRAS-Di Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Fe is an essential element for most living organisms, coordinating with many enzymes responsible for electron transfer and redox reactions to produce energy, mainly through photosynthesis and respiration (Kermeur et al. 2023 ). Fe deficiencies are among the most prevalent micronutrient deficiencies worldwide, affecting 2 billion people and causing more than 0.8 million deaths annually (Murgia et al. 2012 ; WHO 2019). Biofortification is a cost-effective and sustainable approach that aims to solve this problem by boosting the micronutrient content of crops (Connorton and Balk 2019 ). Rice ( Oryza sativa ) is a staple food for over 3.5 billion people and comprises approximately 23% of the calories consumed worldwide (Khush 2003 ; Hackl et al. 2019 ; Kermeur et al. 2023 ). The development of Fe biofortified rice is a solution to the global problem of Fe deficiency anemia (Hackl et al. 2019 ). Rice plants acquire Fe from soil as ferrous Fe (Fe 2+ ) through OsIRT1, OsIRT2, and OsNRAMP1 (Ishimaru et al. 2006 ), and as the phytosiderophore 2′-deoxymugineic acid (DMA)–Fe 3+ complex (Takagi 1976 ; Curie et al. 2000 ) through Yellow Stripe 1 (YS1)/YSL transporters, such as OsYSL15 (Curie et al. 2001 , 2009 ; Inoue et al. 2008 , 2009 ; Lee and An 2009; Suzuki et al. 2021 ). DMA synthesis in rice is catalyzed by nicotianamine synthase (NAS) to produce the Fe 2+ chelator nicotianamine (NA) from three molecules of methionine (Higuchi et al. 1999 , 2001 ), followed by the conversion of NA to DMA via nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) (Takahashi et al. 1999 , 2001 ; Cheng et al. 2007 ; Inoue et al. 2008 ; Bashir et al. 2017 ). A transporter of mugineic acid family phytosiderophores (TOM1) is responsible for transporting DMA from the roots into the rhizosphere (Nozoye et al. 2011 ). Almost all the genes involved in above Fe uptake mechanism are strongly upregulated in response to Fe deficiency (Kobayashi et al. 2014 ). After Fe absorption, OsYSL2 and other YSLs support the long-distance translocation of Fe as MA–Fe 3+ and NA–Fe 2+ complexes from the roots to shoots (Ishimaru et al. 2010 ; Koike et al. 2004 ; Aoyama et al., 2009 ; Senoura et al. 2017 ). Additionally, FERRIC REDUCTASE DEFECTIVE LIKE 1 (OsFRDL1) is a transporter involved in the efflux of citrate into the xylem for efficient Fe translocation (Ariga et al., 2014 ; Yokosho et al. 2016 ). Other transporters are also involved in Fe transport/translocation: OsPEZ1/2, which facilitates Fe transport by effluxing phenolic compounds with Fe-chelating activity (Bashir et al. 2011a ; Ishimaru et al. 2011a b ), EFFLUX TRANSPORTER OF NA 1/2 (OsENA1/2); and OsTOMs NA efflux transporters (Nozoye et al. 2011 , 2019 ). Fe transported into the cell is further distributed to organelles by the vacuolar Fe transporter OsVIT1/2 (Zhang et al. 2012 ; Che et al. 2021 ) and the mitochondrial Fe transporter MIT1 (Bashir et al. 2011b ). The vacuolar MA transporter, OsVMT, also plays a key role in controlling the subcellular partitioning of MAs, thereby regulating metal translocation to grains (Che et al. 2019 ). Excess Fe within cells is sequestered in the vacuoles and the Fe-storage protein ferritin (OsFER1 and OsFER2) (Stein et al. 2009 ; Briat et al. 2010 ). Attempts have been made to generate Fe-enhanced rice plants by controlling the expression of individual genes or a combination of genes responsible for the transport, translocation, and accumulation of Fe using genetic engineering techniques (Masuda et al. 2020 ; Kawakami and Bhullar 2021 ). In addition to these Fe transport- and storage-related genes, manipulation of the transcriptional and post-translational regulators of Fe accumulation could potentially be utilized for Fe biofortification. For example, overexpression of the transcription factors IDEF1 and IRON-REGULATED TRANSCRIPTION FACTOR 2 (OsIRO2) or IRON-REGULATED TRANSCRIPTION FACTOR 3 (OsIRO3) knockout enhances Fe-deficiency responses and increases Fe acquisition. Here, the transcription factors IDEF1 and IDEF2, located upstream of the Fe signaling pathway in rice, positively regulate the early Fe-deficiency response, primarily for most genes involved in DMA–Fe (III) and Fe 2+ uptake (Kobayashi et al. 2007 ; Ogo et al. 2008 ). The basic helix-loop-helix (bHLH) transcription factor OsIRO2 positively regulates Fe deficiency–induced gene expression under the control of IDEF1. The bHLH gene OsIRO3, a homolog of POPYE (PYE) in Arabidopsis, negatively regulates Fe deficiency–inducible genes (Ogo et al. 2007 , 2011 ; Zheng et al. 2010 ; Wang et al. 2020 ). As post-translational regulators, the hemerythrin motif–containing RING zinc-finger proteins (HRZ1 and HRZ2), which function as E3-ubiquitin ligases and homologs of BRUTUS (BTS) in Arabidopsis, negatively control most known Fe deficiency–inducible genes, including the bHLH transcription factors OsIRO2 and OsIRO3, by degradation via the 26S proteasome. Notably, HRZs contain histidine, histidine, and glutamic acid (HHE) domains with direct Fe- and Zn-binding properties and are thus considered promising candidates for Fe sensors in rice cells (Kobayashi et al. 2013 , 2014 , 2022 ; Pullin et al. 2025 ). HRZs also negatively regulate the expression of OsIMA1 and OsIMA2, which are rice orthologs of the small signaling peptide IRON MAN (IMA)/Fe UPTAKE INDUCING PEPTIDE (FEP) found in Arabidopsis (Grillet et al. 2018 ), at the transcriptional level via an unknown pathway (Kobayashi et al. 2021 ). HRZ2 knockdown reportedly enhances Fe deficiency tolerance and Fe accumulation (Kobayashi et al. 2013 , 2022 ). Regarding HRZ1, detailed phenotypic analysis of the previous hrz1 mutant had not been advanced due to its poor growth phenotype. Recently, several transformants and genome-edited rice plants that accumulate the mutated HRZ1 protein lacking the C-terminal region have been created using a transgenic strategy and a genome-editing technique, and exhibited enhanced Fe-deficiency responses, similar to the suppression of HRZ2 (Shinkawa et al. 2025 ). However, since all of these have undergone genetic modification, their yield potential and practicality for use as Fe-biofortified rice have not been examined. In this study, we developed practical Fe-biofortified rice lines by isolating a rice mutant with high Fe content in the shoot from a screening of 3,000 MNU-mutagenized mutant lines. We identified a novel nonsense mutation in the fourth exon of the HRZ1 gene, which corresponds to the beginning of the second hemerythrin domain of HRZ1 proteins. This mutation exhibited a pronounced phenotype characterized by Fe accumulation throughout the shoot, including in leaves and grains. This is the first report to demonstrate that the discovered HRZ1 mutation is beneficial for producing non-transgenic, practical, biofortified rice plants. Results Isolation of a rice mutant tetsu with high Fe content in shoots and xylem sap A rice mutant that accumulated high amounts of Fe was screened from an MNU-mutagenized T65 mutant population (TCM lines). As part of the primary screening, we performed an ionome analysis of the shoots and xylem sap of 21-day-old seedlings of the mutant population, as previously reported (Tanaka et al. 2018 ). Of the 2,704 germinated TCM lines, one mutant line, TCM1587, had higher Fe concentrations in both the shoot and xylem sap (9- and 6.5-fold on average, respectively) than wild-type T65 (Fig. 1 a, b). As a secondary screening step, prompted by the large variation in Fe content within TCM1587, we selected one progeny line with a fixed high Fe accumulation trait, showing 3- and 4-fold increases in shoot Fe and Mn, respectively (Fig. 1 c, d). Zn and Cu concentrations in shoots (Fig. 1 e, f) and Fe, Mn, Zn, and Cu concentrations in roots (Fig. 1 g–j) were not significantly different between T65 and the progeny line. Hereafter, we refer to this line as Transporting Errors in TranSition metal Uptake (tetsu) : tetsu means iron in Japanese. tetsu mutant does not absorb excess toxic heavy metals Because the tetsu mutant accumulated higher concentrations of Fe and Mn than did T65, we assumed an increase in the accumulation of other heavy metals such as Ni, Co, Cd, and Pb. To clarify this, the germinated seeds of this mutant were grown in soil containing low concentrations of Ni, Co, Cd, and Pb (10 mg kg soil − 1 for Ni and 1 mg kg soil − 1 for Co, Cd, and Pb), and the amount of heavy metal absorption was measured (Fig. S1 a, b). The results showed a 1.8-fold increase in the essential element Ni in the shoots and roots of the tetsu mutant compared with that in T65, which was mostly stored in the roots and minimally transferred to the shoots (Fig. S1 c, d). Other non-essential heavy metals (Co, Cd, and Pb) accumulated equally in the tetsu mutant and T65, both in the roots and shoots (Fig. S1 e–g), confirming that tetsu did not excessively accumulate these heavy metals. tetsu mutant accumulates Fe in grains Next, we measured the metal content of brown and polished rice. In the brown rice of the tetsu mutant, Fe increased more than 2-fold, Zn and Cu also increased significantly by over 40%, whereas Mn decreased significantly compared with that in T65 (Fig. 2 a–d). In polished rice, the tetsu mutant exhibited approximately 2-fold higher Fe, 1.3-fold higher Zn, and 1.5-fold higher Cu than those in T65 (Fig. 2 e–h). Because we prepared polished rice at the same milling ratio of 90% between T65 and tetsu , the amount of remaining bran did not affect this difference (Fig. 2 i). Thus, we concluded that tetsu had significantly higher levels of Fe and moderately higher levels of Zn and Cu in the endosperm than did T65. To further verify the high Fe accumulation traits in grains, we analyzed Fe localization in rice grains using Perls’ staining. The stained images visually revealed that Fe was not limited to the outer layers (pericarp and aleurone layer) and scutellum, but also accumulated in the endosperm of the tetsu mutant compared with that in T65 (Fig. 2 j). tetsu mutant has significant alkaline soil tolerance High Fe accumulation in the shoots of the tetsu mutant was also expected to contribute to Fe deficiency tolerance. To confirm this possibility, Fe-deficiency tolerance was investigated in a calcareous alkaline soil environment using culture soil with calcium hydroxide [Ca(OH) 2 ]. At a concentration of 1.5% (w/w) Ca(OH) 2 in the rice culture soil (pH 5.0), soil pH increased to 8.7, and the amount of available Fe decreased to approximately 30% (Fig. 3 a and Fig. S2), which is typical for calcareous Fe-deficient soils. The high pH persisted throughout cultivation; therefore, Ca(OH) 2 was only mixed before planting. A high Fe-containing F 3 population backcrossed with the tetsu mutant to T65 was used to exclude the effects of mutations not involved in Fe accumulation in the tetsu mutant. After 38 days of pre-cultivation in pH 5.0 control soil, plants were transplanted into pH 5.0 or 8.7 soil for an additional 101 days. At pH 5.0, the appearance (Fig. 3 b), SPAD value of the latest expanded leaves (Fig. 3 c), shoot weight (Fig. 3 d), and effective tillering rate (percentage of tillers that led to panicle formation) (Fig. 3 e) were similar between WT and tetsu homozygous plants with no significant difference. Conversely, at pH 8.7, the WT showed significantly lower SPAD values in young leaves than did tetsu at 37 days after transplanting, and although transient recovery was observed, the SPAD values of WT continued to decrease until the end of cultivation (Fig. 3 c). Meanwhile, tetsu maintained young leaf color at the same level as that at pH 5.0 during the cultivation period (Fig. 3 c). Growth at pH 8.7 was equally reduced in both WT and tetsu compared with that at pH 5.0 under alkaline conditions (Fig. 3 a, d), but the effective tillering rate was significantly higher than that in WT at pH 8.7 (Fig. 3 e). At the end of cultivation (101 days after transplanting), seeds in WT were still green and immature, whereas many of the seeds in the tetsu mutant had almost reached full maturity (Fig. 3 f). The Fe concentration in the shoots and brown rice was significantly higher in the tetsu mutant than that in WT plants (Fig. 3 g, h). Since both the WT and tetsu mutant plants had few seeds at pH 8.7, accurate analysis of yield components was not possible; therefore, we conducted a second trial to confirm the above results under milder alkaline conditions using 1.0% Ca(OH) 2 , where the soil pH was approximately 8.0, with the DTPA-Fe level significantly unchanged at the beginning of cultivation compared with that at pH 5.0 (Fig. S2). As shown in Fig. S3a, the grain weight of the tetsu mutant did not change between pH 5.0 and 8.0, whereas that of the WT significantly decreased to approximately 60% at pH 8.0. Total straw weight and effective tillering rate remained unchanged (pH 8.0) in the tetsu mutant, but were decreased in WT at pH 8.0 (Fig. S3b, c). The number of panicles showed no significant difference; however, that in WT tended to decrease at high pH, whereas the tetsu mutant showed no change (Fig. S3d). WT and the tetsu mutant did not significantly differ in the number of tillers, grain weight per panicle, and grain-filling rate (Fig. S3d–h). Taken together, we confirmed that the tetsu mutant was tolerant to alkaline Fe-deficient conditions, maintaining source size (total straw weight) and effective tillering rate, resulting in higher yield in the tetsu mutant than in the WT under these conditions. High Fe accumulation trait in tetsu is associated with a novel nonsense mutation in HRZ1 Next, to identify the gene responsible for the high Fe accumulation of tetsu , we crossed the the tetsu mutant with the Japanese glutinous black–purple rice cultivar ‘Asamurasaki’ with normal Fe and high functional compounds in grains (Pereira-Caro et al. 2013 ; Oo et al. 2025 ) to obtain an F 2 population in which phenotypic segregation could be easily identified (Fig. 4 a). A total of 219 F 2 plants were grown in soil for 16–18 days after germination, and the Fe concentration of the whole shoot was measured. A total of 166 F 2 individuals had Fe concentrations similar to those of ‘Asamurasaki,’ while 53 F 2 individuals had Fe concentrations as high as those of the tetsu mutant (Fig. 4 b). The chi-square test did not reject the null hypothesis of a 3:1 segregation ratio, which is consistent with the idea that the causal gene was single and recessive (Fig. 4 b). To identify the causal gene, the genotypes of F 2 individuals were determined using a recently developed GRAS-Di system (Hosoya et al. 2019 ; Miki et al. 2020 ; Fekih et al. 2023 ), a method based on comprehensive amplicon sequencing. First, a comparison of the amplicons of the parents ( tetsu and ‘Asamurasaki’) revealed 2,090 amplicon markers. These DNA markers mapped widely and uniformly to all rice chromosomes, confirming that a set of good-quality markers had been obtained to identify the causal gene (Fig. 4 c and Fig. S4). Next, we developed an “amplicon index” that quantified the detection or non-detection of each marker in individual F 2 plants. The Δ (amplicon index) was calculated to identify regions where marker detection rates differed significantly between F 2 populations with low and high Fe (Fig. S5). This method successfully narrowed down the causative locus to the 24.3- to 30.3-Mb region of chromosome 1 (Fig. 4 d). To identify all the mutation sites of tetsu within this causative locus, we compared the whole genome sequences of T65 and tetsu using high-precision next-generation sequencing analysis and identified 39 mutation sites. These were all GC/AT substitutions, a typical type of mutation known to occur after MNU mutagenesis. Among these, we found a novel nonsense mutation within the fourth exon of the HRZ1 gene, which encodes a ubiquitin ligase predicted to function as a negative regulator of Fe-uptake/translocation-related genes (Fig. 4 e, f). Other mutation sites included missense mutations in non-coding and untranslated regions, as well as putative genes with no significant reported function, none of which were potential candidates (Fig. 4 e and Table S1 ). Shoot Fe content is increased by the tetsu -type HRZ1 mutation The mutation found in HRZ1 is presumed to express a truncated HRZ1 protein that has only the first HHE domain without the other two HHE domains and the C-terminal zinc-finger domains (Fig. 5 a). The protein that can be produced by such a mutation is similar to the rice hemerythrin domain protein HORZ1(Fig. 5 a), which positively regulates the Fe-deficiency response—the opposite function to the normal HRZs (Kobayashi et al. 2013 ; Shinkawa et al. 2025 ). We developed a dCAPS marker that could efficiently detect this genetic mutation using modified forward primers and the HincII enzyme (Fig. 5 b and Fig. S6). This dCAPS assay reliably detected the HRZ1 gene as a 309 bp fragment in WT, a 261 bp fragment in the homozygous mutant, and both fragments in the heterozygous mutant (Fig. 5 b). Investigating the relationship between HRZ1 genotype and shoot Fe concentration in 114 F 2 plants from tetsu בAsamurasaki’ discriminated by this dCAPS marker, we found that the homozygous mutant of HRZ1 had significantly higher Fe content than did WT or heterozygous plants (Fig. 5 c). This result suggests that the high Fe accumulation in the tetsu mutant was caused by a tetsu -type nonsense mutation in HRZ1 . tetsu(hrz1) mutant strongly induces an Fe-deficiency response at the transcriptional level Previously, RNAi-knockdown lines of HRZ2 showed increased expression of various Fe uptake- and utilization-related genes in Fe-sufficient roots, similar to Fe-deficient non-transformant rice roots (Kobayashi et al. 2013 ). To confirm whether the tetsu ( hrz1 ) mutant also upregulated the expression of such genes, we performed RNA-seq analysis in WT and the tetsu ( hrz1 ) mutant grown in normal Fe-sufficient soil (Fig. 3 ). Because high Fe accumulation occurred in the shoots of tetsu ( hrz1 ) plants (Fig. 1 ), we investigated its expression in fully developed young leaves. As shown in Table 1 , a significant increase in the expression of various Fe deficiency–induced genes was found in the tetsu ( hrz1 ) mutant compared with that in WT, including IMA1 , IMA2 , IRO2, IRO3 , NAS1, NAS2, YSL2 , NRAMP1 , TOM1 , OPT7 , and PTR (a putative NA/DMA efflux transporter). HHE domain–containing proteins ( HRZs and HORZ1 ) and IDEF1, which control Fe deficiency signals further upstream, were not significantly altered in the mutant. A significant, but very slight, decrease in IDEF2 expression was observed in the tetsu ( hrz1 ) mutant. NAS3 levels declined, consistent with previous reports that NAS3 was repressed by Fe deficiency but induced by excess Fe (Aung et al. 2018 ). In addition, the expression of the ferritin genes FER1 and FER2 , which are known to be induced by excess Fe, was downregulated in the leaves of tetsu ( hrz1 ). These results clearly indicate that the tetsu ( hrz1 ) mutant enhanced the Fe-deficiency responses but suppressed the excess Fe responses in the shoot despite containing elevated Fe concentrations. No upregulation of any of the genes encoding proteins involved in the transport and regulation of other heavy metals or metal-chelating compounds (phenolics and citrate) was observed in the tetsu ( hrz1 ) mutant (Tables 1 and 2 ). Table 1 RNA-seq analysis of genes involved in Fe-deficiency responses, DMA synthesis/secretion, Fe transport/translocation, and Fe sequestration/storage in leaves of the wild-type and tetsu mutant Transcript ID Gene Description WT tetsu ( hrz1 ) Fold change* p -value transcripts per million (TPM) Hemerythrin domain-containing genes Os01t0689451-01 HRZ1 Hemerythrin motif-containing RING-& Zinc-finger protein 1 1.02 ± 0.22 0.787 ± 0.11 0.771 0.4 Os05t0551000-01 HRZ2 Hemerythrin motif-containing RING-& Zinc-finger protein 2 1.66 ± 0.37 1.87 ± 0.33 1.12 0.7 Os01t0861700-00 HORZ1 Hemerythrin motif-containing protein without RING- and Zn-finger 1 0.402 ± 0.21 0.175 ± 0.16 0.435 0.44 Constitutively expressed Fe-related transcription factors Os08t0101000-01 IDEF1 Iron deficiency-responsive cis-acting element binding factor 1 13.1 ± 1.81 11.1 ± 1.02 0.851 0.40 Os05t0426200-02 IDEF2 Iron deficiency-responsive cis-acting element binding factor 2 58.7 ± 1.40 49.6 ± 2.38 0.845 0.03 * Fe deficiency-induced Fe-related transcriptional and signaling regulators Os01t0647200-01 IMA1 Fe-deficiency-inducible IRON MAN 1 3.24 ± 1.05 613 ± 115 189 0.0061 ** Os01t0647200-04 IMA1 12.6 ± 3.80 2170 ± 525 173 0.015 * Os07t0142100-01 IMA2 Fe-deficiency-inducible IRON MAN 2 0.0967 ± 0.10 2340 ± 725 > 10,000 0.032 * Os01t0952800-01 IRO2 Iron-related transcription factor 2, bHLH056 0.00433 ± 0.00 157 ± 21.5 > 10,000 0.0019 ** Os03t0379300-01 IRO3 Iron-related transcription factor 3, bHLH063 3.62 ± 0.62 22.8 ± 1.58 6.3 0.0004 *** Os03t0379300-02 IRO3 1.31 ± 0.84 15.7 ± 2.34 11.9 0.0044 ** Os03t0379300-03 IRO3 0.112 ± 0.09 1.16 ± 0.31 10.4 0.031 * Biosynthesis of mugineic acid family phytosiderophores Os03t0307300-01 NAS1 Nicotianamine synthase 1 0.0118 ± 0.01 2.09 ± 0.28 177 0.0018 ** Os03t0307200-01 NAS2 Nicotianamine synthase 2 0.114 ± 0.11 4.45 ± 0.86 38.9 0.0074 ** Os07t0689600-01 NAS3 Nicotianamine synthase 3 1.96 ± 0.32 0.721 ± 0.31 0.367 0.048 * Os02t0306401-01 NAAT1 Nicotianamine amino-transferase 1 35.8 ± 7.39 15.7 ± 3.61 0.439 0.071 Os03t0237100-01 DMAS1 Deoxymugineic acid synthase 1 17.4 ± 1.54 24.1 ± 3.94 1.390 0.184 Fe uptake and/or translocation Os02t0649900-01 YSL2 Fe- and Mn-nicotianamine transporter 0.0424 ± 0.04 22 ± 4.09 519 0.0058 ** Os07t0258400-01 NRAMP1 Homologues of mammalian Nramp1, Fe transporter 0.0514 ± 0.05 36.9 ± 6.75 718 0.0055 ** Os07t0258400-02 NRAMP1 N.D. 14.9 ± 2.42 > 10,000 0.0036 ** Os07t0258400-03 NRAMP1 N.D. 3.69 ± 1.64 > 10,000 0.088 Os11t0134900-01 TOM1 Efflux transporter of mugineic acids N.D. 0.528 ± 0.04 > 10,000 0.0002 *** Os03t0751100-01 OPT7 Oligopeptide Transporter 7 9.02 ± 1.12 15.8 ± 1.14 1.75 0.013 * Os03t0751100-02 OPT7 6.25 ± 1.32 13.1 ± 0.52 2.09 0.0084 ** Os01t0871500-01 PTR Peptide transporter (Arabidopsis NAET-like) 44.3 ± 1.12 226 ± 25.3 5.11 0.002 ** Os03t0667500-01 IRT1 Iron-regulated transporter 1 2.23 ± 0.73 2.11 ± 0.49 0.947 0.9 Os03t0667300-01 IRT2 Iron-regulated transporter 2 N.D. 0.0375 ± 0.03 > 10,000 0.21 Os11t0151500-01 ENA1 Efflux of nicotianamine 1 0.00498 ± 0.00 0.343 ± 0.13 68.9 0.06 Os06t0695800-01 ENA2 Efflux of nicotianamine 2 0.630 ± 0.11 0.570 ± 0.20 0.906 0.81 Os12t0282000-01 MIR Mitochondrial iron-regulated gene N.D. 0.288 ± 0.18 > 10,000 0.18 Os04t0463400-01 VIT1 Vacuolar Iron Transporter 1 15.5 ± 0.75 18.5 ± 1.44 1.19 0.14 Os09t0396900-02 VIT2 Vacuolar Iron Transporter 2 6.41 ± 2.10 0.783 ± 0.19 0.122 0.056 Os11t0106700-01 FER1 Ferritin 1 25.9 ± 2.58 15.1 ± 0.71 0.584 0.016 * Os11t0106700-02 FER1 244 ± 27.8 131 ± 19.3 0.536 0.029 * Os12t0106000-01 FER2 Ferritin 2 296 ± 39.4 151 ± 21.4 0.512 0.032 * * Fold-change indicates the expression ratio of tetsu /WT. Bold numbers indicate genes in the tetsu mutant that were significantly upregulated, whereas underlined numbers indicate genes that were significantly downregulated. WT and tetsu homozygotes were isolated from the F 2 population obtained after a single backcross to T65 for the tetsu mutant, with three biological replicates ( n = 3). Plants were grown under normal soil conditions for 60 days at 26°C in a greenhouse. Young leaves (6th leaf) were harvested at 2:30 p.m. Data are expressed as transcripts per million. Significant differences (* P < 0.05, ** P < 0.01, *** P < 0.001) between the WT and tetsu mutants were tested using the Student's t-test. Table 2 RNA-seq analysis of transporters related to heavy metals in leaves of the wild-type and tetsu mutant Transcript ID Gene Description WT tetsu ( hrz1 ) Fold change* p -value transcripts per million (TPM) Other transporters related to translocation of Fe or other metals Os07t0232800-01 ZIP8 Zinc transporter 8 96.7 ± 14.4 32.9 ± 10.4 0.340 0.023 * Os07t0232800-02 ZIP8 Zinc transporter 8 4.69 ± 0.41 2.01 ± 0.82 0.429 0.043 * Os03t0571900-01 PEZ1 Phenolics efflux zero 1 6.48 ± 0.54 4.45 ± 1.46 0.686 0.26 Os03t0572900-01 PEZ2 Phenolics efflux zero 2 34 ± 0.90 30.2 ± 5.12 0.888 0.51 Os12t0133100-02 VMT Vacuolar mugineic acid transporter (ZIFL12) 16.5 ± 2.50 16.3 ± 1.22 0.986 0.937 Os03t0216700-01 FRDL1 Citrate transporter (Fe-citrate transport) 1.86 ± 0.29 1.72 ± 0.72 0.927 0.870 Os10t0206800-01 FRDL2 Citrate transporter (Al-induced) 14.3 ± 0.67 10.43 ± 1.88 0.731 0.128 Os10t0206800-02 FRDL2 Citrate transporter (Al-induced) 6.57 ± 0.58 4.99 ± 0.80 0.759 0.183 Os02t0833100-01 FRDL3 Citrate transporter, putative 1.09 ± 0.09 1.56 ± 0.28 1.43 0.185 Os01t0919100-01 FRDL4 Citrate transporter (Al-induced) N.D. N.D. - - Os02t0650300-01 YSL15 Fe- and Mn-nicotianamine transporter N.D. N.D. - - Os06t0560000-02 FPN1 Ferroportin, intracellular Ni and Co transporter (IREG1) 2.55 ± 0.22 2.99 ± 0.45 1.17 0.424 Os07t0257200-01 NRAMP5 Low cadmium accumulation 1 0.580 ± 0.31 0.613 ± 0.31 1.06 0.940 Os07t0232900-01 HMA3 Heavy metal ATPase 3 0.801 ± 0.25 0.973 ± 0.24 1.22 0.640 * Fold-change indicates the expression ratio of tetsu /WT. Underlined numbers indicate genes that were significantly downregulated in the tetsu mutant. The expression values were obtained from the same dataset, as shown in Table 1 . First practical attempt to increase Fe content in rice grain while simultaneously increasing antioxidant components Sterility occasionally occurred in the original TCM1587 mutant line used for selecting the tetsu ( hrz1 ) mutant, independent of high Fe accumulation (Fig. 6 a). This is due to the vast number of genomic mutations that occur randomly in the MNU mutagen of the original tetsu ( hrz1 ) genome. Therefore, we further screened lines with improved defective traits from the progeny of the F 2 population ( tetsu × ‘Asamurasaki’). After 10 generations of repeated selfing, we obtained pure recombinant inbred lines (RILs) (Fig. 6 b). Among the RILs, A-5BM, C-1M, and A-3U showed normal growth and fertility comparable to those of T65 and ‘Asamurasaki’ (Fig. 6 c,d). Because the parent cultivar ‘Asamurasaki’ is a glutinous (waxy) black–purple rice that accumulates high concentrations of many functional components and antioxidants in its grains (Pereira-Caro et al., 2013 ), the selected RILs inherited these traits in various combinations, i.e., A-5BM, which is black–purple rice and glutinous (waxy), C-1M, which is white rice and glutinous (waxy), and A-3U, which is white rice that is not glutinous (non-waxy) (Fig. 6 e). Using the dCAPS assay, we confirmed that the three RILs harbored a tetsu ( hrz1 ) mutation (Fig. 6 f). Compared with WT T65 and ‘Asamurasaki,’ the number of grains per spikelet when grown in the normal soil was significantly lower for the original tetsu ( hrz1 ) mutant, but comparable for the three RILs (Figs. 7 a). The total grain weight was also significantly improved in the three RILs compared with that in the tetsu ( hrz1 ) mutant (Fig. 7 b). Shoot Fe concentrations in the three RILs remained significantly increased by approximately 3-fold compared with those in WT cultivars (T65 and ‘Asamurasaki’) (Fig. 7 c). Fe concentrations in brown rice were also up to 2-fold higher in these RILs than in WT cultivars (Figs. 7 d). Fe concentrations in polished rice also showed an increasing trend, averaging 3- to 5-fold higher compared with those in ‘Asamurasaki’ and 1.6- to 2.7-fold higher than those in T65 (Fig. 7 e). Thus, RILs that regained normal reproductive growth through crossing with ‘Asamurasaki’ maintained stable, high Fe contents across generations, demonstrating that the tetsu ( hrz1 ) mutation itself is not a harmful mutation that reduces yield potential. Among these RILs, A-5BM had significantly more antioxidant components, such as anthocyanins and various phenolic acids, which are abundant in ‘Asamurasaki’ (Fig. 7 f–i). These results indicate that the identified novel HRZ1 mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars with various beneficial traits. Discussion We identified a new nonsense mutation in HRZ1 (Figs. 4 and 5 ) in the Fe-accumulating mutant tetsu . Notably, RILs with the tetsu(hrz1) mutation (Figs. 6 and 7 ) or the backcross line of the tetsu ( hrz1 ) mutant (Fig. S3) did not show abnormalities in growth or fertility, with an extremely high Fe content in both the leaves and seeds. This contrasts with the previously reported hrz1-1 mutant (Kobayashi et al. 2013 ) or hrz1-2 genome editing line (Zhang et al. 2017 ), both of which are reportedly sterile and/or have poor growth. Because of these undesirable characteristics, achieving Fe biofortification using the previously reported HRZ knockout/knockdown lines has been challenging. More recently, Shinkawa et al. ( 2025 ) created rice plants with mutated HRZ1 using a transgenic strategy and CRISPR/Cas9 genome editing techniques. These transformants/mutants targeting HRZ1 exhibited Fe deficiency tolerance and increased Fe content in brown rice, similar to the tetsu ( hrz1 ) mutant. However, the shoot Fe content, yield potential, and sterility of these transformants/mutants have not been reported in detail. Among the hrz1 mutants reported to date, hrz1-1 showed little or no change in shoot Fe compared with that in WT (Kobayashi et al. 2013 ). This contradicts the tetsu(hrz1) mutant, which accumulated more than 3-fold the amount of Fe in the leaves than in the shoots (Fig. 1 c, 5 c, 7 a). The factor that caused differences between the previously reported hrz1 mutants and the current tetsu(hrz1) mutant is likely the difference in the mutation site. A previous hrz1-1 had a DNA insertion upstream of the 5'-UTR, causing HRZ1 expression to be moderately suppressed. In contrast, the tetsu(hrz1) mutant has a nonsense mutation caused by a single-base substitution at the beginning of the second HHE domain, which translates into a truncated HRZ1 with only one complete HHE domain. The shortened structure of the HRZ1 protein was similar to that of another HHE-containing protein, HORZ1 (Fig. 5 a), which functions as a positive regulator of Fe-deficiency responses. Thus, one possibility is that mutated HRZ1, which may accumulate in the tetsu(hrz1) mutant, could function as a positive regulator of Fe-deficiency responses in this mutant. This hypothesis is supported by the findings of Shinkawa et al. ( 2025 ), which suggest that overexpression of GFP-HRZ1ΔRZ constructs, comprising only two hemerythrin domains of HRZ1 without the other domains, enhances Fe deficiency responses in a dominant-negative manner due to the mutated shortened HRZ1 proteins. Further research is required to confirm our hypothesis regarding the tetsu ( hrz1 ) mutation. Among the genes upregulated in the tetsu(hrz1) mutant (Table 1 ), IMA1 , IMA 2, IRO2 , and IRO3 were significantly upregulated at the transcriptional level in shoots. In addition, Fe deficiency–inducible genes such as YSL2 , TOM1 , ENA1 , NRAMP1 , and NAS1/2 were strongly upregulated in the shoots of the tetsu(hrz1) mutant. Earlier reports have shown that suppressing HRZ1/2 expression using RNAi causes high expression of these Fe-deficiency response genes (Kobayashi et al. 2014 ). Thus, the ability of the tetsu(hrz1) mutant to accumulate high levels of Fe in the shoots is assumed to be the reason for the enhanced Fe-deficiency responses. Furthermore, the tetsu(hrz1) mutant exhibited a 5-fold increase in the putative NA/DMA efflux transporter PTR (Os01g0871500) (Nozoye et al. 2011 ) and a 2-fold increase in the expression of OPT7 (Table 1 ). The gene with high homology to PTR is NAET 1/2 in Arabidopsis, which is responsible for Fe source-to-sink transport (Chao et al., 2021 ). OPT7 is a transporter responsible for the xylem unloading of Fe 2+ and the preferential distribution of Fe in developing tissues (Bashir et al. 2015 ; Yamaji et al. 2024 ). Thus, the increased expression of PTR and OPT7 in the tetsu(hrz1) mutant could contribute to the marked increase in Fe concentration in the grains (Fig. 1 ). In addition to Fe, the tetsu(hrz1) mutant accumulated high Mn and Ni levels in the shoots (Fig. 1 and Fig. S1 ) and Zn and Cu in the grains (Figs. 4 and 5 ). This can be explained by the fact that DMA and NA not only bind to Fe but also have an affinity for Mn 2+ , Ni 2+ , Cu 2+ , and Zn 2+ , thereby facilitating the translocation and transport of these essential transition metals in shoots (Murakami et al. 1989 ; Mari et al. 2006 ; Curie et al. 2009 ). In contrast, the contents of non-essential Cd, Pb, and Co were not increased in the shoots of the tetsu(hrz1) mutant (Fig. S1 ). Although recent reports indicate that the overexpression of NAS in yeasts or Arabidopsis enhances Cd mobility (Hollmann et al., 2025 ), we did not obtain any data indicating that the hrz1 mutation enhances Cd uptake. This is likely because the primary Cd uptake pathway in rice is OsNRAMP5, which transports Cd²⁺, the predominant chemical form in aerobic paddy fields (Ishikawa et al. 2012 ; Sasaki et al. 2012 ). Furthermore, we confirmed the absence of increased expression of any transporters associated with heavy metal transport in the tetsu(hrz1) mutant (Table 2 ), including OsZIP s (Zn and Cd transporters), OsNramp5 (Cd, Pb, and Mn transporters), OsHMA3 (vacuolar Cd transporter), and FPN1 (Ni and Co transporters; Kaur et al. 2021 ; Kan et al. 2022 ). These results revealed that the tetsu ( hrz1 ) mutation could serve as a beneficial target gene site for improvement in Fe biofortification, simultaneously increasing the Zn and Cu content in grains while preventing the absorption of harmful elements. When developing Fe-biofortified crops using hrz / bts mutations, considering the risk of Fe toxicity in plants is essential. Unlike rice, most other plant species are sensitive to excess Fe. Indeed, severe Fe toxicity with defective embryo development and leaf necrosis could occur because of increased amounts of Fe in the shoots of Arabidopsis bts mutants and a pea HRZs/BTS ortholog mutant dgl ( degenerate leaves ) grown under normal soil conditions (Welch and Larue 1990 ; Selote et al. 2015 ; Harrington et al. 2024 ). Thus, for crops that are not tolerant to Fe toxicity, it may be challenging to confer a trait that significantly increases Fe content in shoots, such as the tetsu ( hrz1 ) mutant. Previous reports have indicated that hrz1 knockdown rice is more prone to Fe toxicity when grown under Fe-excess conditions (Aung et al., 2018 ; Kobayashi et al., 2019 ). In the present study, although no adverse effects on yield due to excess Fe were observed in RILs with the tetsu ( hrz1 ) mutation under the soil conditions used (Fig. 7 ), bronzing, an indicator of excess Fe, occasionally appeared on the leaf tips during later growth stages. Therefore, enhancing excess Fe tolerance in rice plants harboring the tetsu ( hrz1 ) mutation through breeding or agronomic approaches will contribute to further dissemination and practical applications of Fe-biofortified rice in the future. In this context, recently reported rice lines possessing excess Fe tolerance traits (Rosdianti et al. 2025 ) may be a promising option for breeding materials. Although various attempts have been made to develop Fe-biofortified rice using the transgenic strategy, its widespread commercialization is limited by the difficulty in obtaining approval or acceptance of such genetically modified plants in various countries. In this context, mutation breeding with a DNA marker for the tetsu -type mutation is an effective strategy to facilitate the practical application of Fe biofortification in non-genetically modified rice. Here, we succeeded in conferring rice with increased Fe and other nutritional, polyphenol, and phenolic components (Fig. 7 ). The RILs generated in this study can be used in regular edible rice or processed rice products. In summary, we successfully established Fe-biofortified rice lines that exhibited normal growth, fertility, and various brown rice traits by breeding a tetsu ( hrz1 ) mutant. These results confirmed that the identified novel HRZ1 mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars. Currently, we are conducting safety tests in animals to confirm their efficacy and anticipate that these RILs will become widely available. Materials and Methods Plant materials and growth conditions T65 (accession no. T0504) seeds were provided by the National Institute of Genetics (Japan). The T65 MNU-induced rice mutants (TCM mutants) were generated by T.K. (Kyushu University) and provided to T.F. or A.S. through the National BioResource Project (MEXT, Japan). ‘Asamurasaki’ seeds were purchased from a Japanese seed company (Noguchi Seeds Co., Saitama, Japan). For the primary screening of the TCM mutants, 128-cell plug trays (Takii Seed Co., Kyoto, Japan) were used for cultivation with commercial soil at pH 5.0, N:P:K = 0.8:2.0:1.0 (Kumiai Chemical Industry Co., Tokyo, Japan). The mineral content of the soil was determined as described previously (Uraguchi et al. 2011 ). The seeds were disinfected by soaking in 60°C water for 10 min, followed by watering for 3 days to promote germination. For each seedling tray, eight plants of the T65 and 90 lines of TCM mutants were sown, totaling 2887 TCM lines. The plants were cultivated for 21 days under natural light in a greenhouse. For the cultivation of tetsu mutant selected from the seed pool of TCM1587 was grown on soil in an air-conditioned greenhouse (26 ± 3°C). For yield surveys of the tetsu(hrz1) mutant and RIL lines, plants were grown in soil in a controlled greenhouse under natural light and supplemental light with a light intensity of more than 400 µmol m − 2 s − 1 during the day, with a 14 h light (28°C)/10 h dark (25°C) cycle. For the alkaline soil experiments, young seedlings were pre-cultured in soil (pH 5.0) for 38 days and then transferred to soil with pH 5.0 or 8.7. Xylem sap collection for determining metal concentrations Xylem sap collection was performed according to previous reports (Uraguchi et al. 2011 ; Yamamura et al. 2024 ) with minor modifications. Briefly, the aboveground portion was cut at a point 3 cm above the ground using a razor blade. A 1-mL chip filled with small pieces of quartz wool was placed over the cut surface, and the exuding xylem sap was absorbed into the quartz wool over a 3-hour period. The 1-mL chip containing quartz wool was inverted and placed into a 2-mL tube, and the xylem sap was collected by centrifugation at 3,500 rpm for 2 min. The collected xylem sap was weighed, stored at − 80°C until analysis. Analysis of metal concentration in rice plants In the metal analysis of shoots during primary screening, up to 100 mg of homogenized dry rice leaves was added to a Teflon-coated centrifuge tube, and 3 mL of a mixture of concentrated nitric acid and hydrogen peroxide (3:1) was added. The sample was placed in a heat block and heated at 80°C for 1 h and 100°C for 20 min, then dissolved by a vortex mixer. The reaction was continued at 150°C for 4 h, with the acid mixture added as needed to complete the reaction. After drying, the sample was resuspended in 0.08 N nitric acid containing 2 ppb indium (In) as an internal standard. For secondary screening and subsequent analyses, 50 mg or less of dried rice leaf powder or 5–10 grains of brown rice were dissolved in 5 mL of concentrated nitric acid. The collected xylem sap was diluted with a fixed volume of 1 N nitric acid. The supernatants of the inorganic element extraction solutions were analyzed using inductively coupled plasma mass spectrometry (ICP-MS, model SPQ 9700; SII Nano Technology, currently Hitachi High-Tech Science, Inc.) for the 1st screening and a furnace atomic absorption spectrophotometer (AA-6300 with GFA-EX7i, Shimadzu, Tokyo, Japan) for the 2nd screening as described (Saito et al., 2021 ). Iron staining of rice seeds using Perls’ staining Perls’ staining was performed by adding 1 mL of Perls’ staining solution (4% v/v HCl and 4% w/v K-ferrocyanide) (Roschzttardtz et al. 2009 ) to 7–8 cut brown rice grains, followed by staining for 90 min. After washing with ultrapure water, images were captured using a stereomicroscope. To prepare longitudinal brown rice sections, one side of the brown rice was adhered to the surface of the cap of a 2 mL screw tube using instant adhesive (Aron α, Quick-Setting Multi-Purpose Extra Type, Konishi Co., Ltd., Osaka, Japan). The 2-mL tube cap, with the rice-adhered side facing upward, was then fixed to the sample tray of a microtome (DTK-1000, DOSAKA EM Co., Ltd., Kyoto, Japan) using the same instant adhesive. A razor blade was aligned with the center of the rice germ, and the sample was cut in half using a microtome set to a vibration frequency of 10 Hz (maximum value) and a cutting speed of 2–3 mm/s. The side without the adhesive was used for Perls’ staining. Measurement of SPAD values in leaves The chlorophyll concentration indices of the leaves were measured using a mobile device (SPAD-50-Plus; KONICA MINOLTA JAPAN, Inc., Tokyo, Japan), and the average of three central areas of the latest expanded leaves was measured for each plant. Soil analysis Soil pH was measured by placing 10.0 g of air-dried soil in a 100-mL glass container, adding 100 mL of pure water, and shaking at 25°C and 135 rpm for 24 h until the pH of the alkaline soil solution reached equilibrium. The pH of the supernatant was then measured. Soil-soluble Fe content was determined according to a well-established method (Lindsay and Norvell 1978 ). Briefly, a 20-mL diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) extraction solution (5 mM DTPA, 0.1 M triethanolamine hydrochloride, 10 mM calcium chloride, adjusted to pH 7.3 with NaOH) was added to 10.0 g of air-dried soil. The mixture was shaken at 25°C and 135 rpm for 2 h. The supernatant was filtered through No. 2 filter paper (Advantech Co., Ltd., Tokyo, Japan), and nitric acid was added to achieve a final concentration of 1%. The DTPA-extracted Fe was then measured using furnace atomic absorption spectroscopy (AA-6300 with GFA-EX7i, Shimadzu, Tokyo, Japan). Data collection procedures for GRAS-Di A total of 97 individuals from the F 2 population and their parents were used for GRAS-Di analysis (Toyota Motor Corporation, Tokyo, Japan). First, germinated seeds were grown in soil for 16–18 days, and then the shoots were cut with a razor blade. The Fe content of the shoots was analyzed in advance. After approximately 40 days, new shoots regenerated from the remaining plant stems. Based on the Fe content values, 54 high-Fe F 2 individuals and 50 low-Fe F 2 individuals, or 3 individuals from each parent, were selected. Healthy and most recently expanded leaves were collected, washed, and immediately frozen in liquid nitrogen. The leaves were then ground to a fine powder using a mortar and pestle under liquid nitrogen, and genomic DNA was extracted using a DNeasy Plant Mini Kit (69104; QIAGEN). GRAS-Di libraries were constructed using a NovaSeq 6000 S4 reagent kit. Libraries were sequenced using an Illumina NovaSeq 6000 (Sequence Mode: 2×150; Flow Cell Type: S4; Illumina, San Diego, CA, USA). Genotyping was conducted using 38,950 dominant single-dose markers generated using GRAS-Di software ver. 1.0.5. (TOYOTA, Aichi, Japan). Sequence reads derived from sequence adapters and those with low sequence quality were excluded from data analysis. Among the resulting 14,757 amplicons, those showing clear amplification differences between the parents were selected. As a result, 1,068 amplicons positive only in the tetsu(hrz1) mutant were designated as C1, and 1,022 amplicons positive only in ‘Asamurasaki’ were designated as C2, yielding a total of 2,090 dominant GRAS-Di markers. The markers obtained were used to determine candidate positions in the reference genome sequence. Whole genome sequencing analysis of T65 and tetsu(hrz1) mutant Five WT (T65) and eight tetsu(hrz1) mutant plants exhibiting a high-Fe phenotype were cultivated for 1 month after sowing. Genomic DNA was extracted from young leaves using the DNeasy Plant Mini Kit (69104; QIAGEN). Genomic DNA from each individual was mixed in a single tube to ensure equal amounts, resulting in bulk DNA samples for T65 and the tetsu(hrz1) mutants. Bulk DNA samples were analyzed using a Genomic DNA ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA) to determine the DNA Integrity Number (DIN), which was approximately 7, confirming the high integrity of genomic DNA. Genome shotgun analysis was performed using HiSeq X, with a read length of 2 × 150. Data analysis involved cleaning the reads using Trimmomatic software (ver. 0.39) and mapping them to the reference sequence using BWA (ver. 0.7.17). The reference sequence used was “ Oryza sativa , IRGSP-1.0.” Both samples contained more than 200 Mb of reads, with 99.8% of the reads mapped. Additionally, over 90% of mapped reads covered the reference sequence at 50× depth, yielding high-precision sequence information. Picard tools (ver. 1.111) was used to remove the duplicate reads. Finally, we listed the bases that did not match T65 and the tetsu(hrz1) mutant (Table S1 ). The identified tetsu(hrz1) mutation sites were confirmed using Sanger sequencing. dCAPS method to detect the mutation in HRZ1 gene To detect tetsu -type mutations in HRZ1 , we created primers based on dCAPS Finder 2.0 ( http://helix.wustl.edu/dcaps/dcaps.html ) (Neff et al. 2002 ), setting the value for mismatches to 1. Among the lists of candidate primers, we selected the following primer sets that can cleave the objective PCR products with HincII: 5'-CTA TTG ATG GTC AGG TTG AAA GGC ATC CCA TAG ATG AGA TTC TGT GTT G-3' (49 bp) for the forward primer and 5'-AAC CTG AAT ACA CTA AGA GAA AGG T-3' (25 bp) for the reverse primer. Genomic DNA for PCR was extracted based on conventional methods (Edwards et al. 1991 ). Scissored young rice leaves less than 1 cm in length were crushed with two stainless steel beads in a 2-mL tube, and the supernatant was collected and purified using the isopropanol precipitation method. More detailed information regarding the dCAPS method is provided in Fig. S6. RNA-Seq analysis From the F 3 population that was backcrossed T65 with the tetsu(hrz1) mutant, homozygous WT ( HRZ +/+) and tetsu ( hrz1-/- ) plants were selected. Leaves from two individuals were combined into one sample, and six individuals ( n = 3) were analyzed. The plants were grown in a greenhouse at 26°C under natural light for 60 days after sowing in culture soil (Bonsol No. 1; Sumitomo Chemical, Tokyo, Japan), and the youngest maximum expanded leaves (sixth leaf) of the main culms were used for analysis. Approximately 100 mg of the frozen leaf powder that had been ground in a mortar and pestle was transferred to a 2-mL tube (TM-626; TOMY MEDICO. Ltd., Tokyo, Japan) containing the RNA extraction solution of an RNeasy Plant Mini Kit (74904; QIAGEN), and crushed using two stainless steel beads (SUB-50, 4.8 mm φ; TOMY MEDICO. Ltd.) in a bead-crushing device (Micro Smash TM MS-100; TOMY MEDICO. Ltd.) at 4,000 rpm for 100 s. Genomic DNA was removed through DNase treatment, according to the standard protocol for the RNeasy Plant Mini Kit. cDNA was prepared by targeting RNA with a poly A tail using the SMART-Seq v4 Ultra Low Input Kit (#634888; Clontech Laboratories, Inc., Mountain View, CA, USA). A Nexera XT DNA Library Prep kit (Illumina) was used to create a cDNA library, and at least 80 million reads were sequenced per sample using NovaSeq (Illumina). Data analysis was performed using the DRAGEN Bio-IT Platform (version 3.7.5; Illumina). The read sequences obtained from the sequencing analysis were mapped to the reference genome sequence ( Oryza sativa IRGSP-1.0), and approximately 99% of all reads were identified. The expression levels of the genes and transcripts were calculated based on the positional information obtained from the mapping and gene definition files. Measurement of total anthocyanin in rice grains Anthocyanin content was measured according to Watanabe et al. ( 2014 ). Approximately 100 mg of brown rice was mixed with 1 mL of anthocyanin extraction solvent (methanol: water: trifluoroacetic acid = 40:60:0.5), and the mixture was incubated at 37°C overnight. The mixture was then ground four times using a bead-crushing device (Micro Smash TM MS-100, TOMY MEDICO. Ltd.) with three zirconia beads (φ3 mm) at 4,000 rpm for 120 s, with ice cooling applied each time. Centrifugation was performed at 15,000 × g for 10 min at 4°C, and the supernatant was collected. The supernatant was diluted 5-fold with the extraction solvent, centrifuged at 15,000 × g for 5 min at room temperature to remove insoluble residues, and the absorbance was measured at 525 nm. Total anthocyanin content was calculated based on the molar extinction coefficient of cyanidin-3-glucoside (Singh et al., 2022). Measurement of phenolic acids in rice grains A total of 600 µL methanol containing 50 µM internal standards (L-methionine sulfone, 2-morpholinoethanesulfonic acid, and 13 C 6 -glucose) was added to 45–50 mg of brown rice powder and mixed using a bead-crushing device under cooling conditions (1,500 rpm, 120 s × 2 times). Thereafter, 600 µL of Milli-Q water was added, stirred, and centrifuged (2,300 × g , 4°C, 5 min). The supernatant was transferred to an ultrafiltration tube (UltraFree-MC-PLHCC, 5 kDa ultrafiltration membrane, Human Metabolome Technologies, Inc. Yamagata, Japan), and centrifuged (9,100 × g , 4°C, 120 min). Subsequently, the filtrate was dried, dissolved in an aliquot of Milli-Q water, and used for measurements. The sample was analyzed using an Agilent CE-TOFMS system (Agilent Technologies) with a fused silica capillary (i.d. 50 µm × 80 cm) in anion mode. Peaks detected using CE-TOFMS were automatically extracted using the automatic integration software MasterHands ver.2.19.0.2. The relative area values were calculated by dividing the area of the target peak by the product of the area of the internal standard and sample amount. Based on the m/z and MT values, all substances registered in the metabolite library were compared to identify phenolic compounds. The acceptable error for the search was set to ± 0.5 min for MT and ± 10 ppm for m/z. For the obtained peaks, the relative area value ratios for each group were calculated and t -tests were performed. Statistical analysis Comparisons between two groups were performed using Student’s t-test, and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey’s test. All statistical analyses were conducted using Microsoft Excel and Statistics Kingdom (2017): ANOVA Calculator ( https://www.statskingdom.com/180Anova1way.html ). Abbreviations MNU; N-methyl-N-nitrosourea, T65; Taichung-65, GRAS-Di; Genotyping by Random Amplicon Sequencing-Direct, HRZ1/2; Hemerythrin (or Hemerythrin) motif-containing Ring Zinc-finger protein 1/2, RIL; Recombinant Inbred Line, IRT1/2; Iron-Regulated Transporter 1/2, DMA; 2’-deoxymugineic acid, NAS; Nicotianamine Synthase, NA; nicotianamine, NAAT; Nicotianamine Aminotransferase, DMAS; Deoxymugineic Acid Synthase, TOM1; Transporter of Mugineic acid family phytosiderophores 1, YS1; Yellow Stripe 1 transporter, YSL; YS1-Like transporter, IDS2/3; Iron Deficiency Specific clone no. 2/3 (DMA dioxygenases), IDEF1/2; Iron-Deficiency-responsive Elements-binding transcription Factor 1/2, OsFRDL1; Ferric Reductase Defective Like 1, PEZ1/2; Phenolics Efflux Transporter 1/2, ENA; Efflux transporter of NA, VMT; Vacuolar Mugineic acid Transporter, VIT1/2; Vacuolar Iron Transporter 1/2, FER; Fe-storage protein ferritin, MIT; Mitochondrial Iron Transporter, OsIRO2/3; Iron-regulated transcription factor 2/3, bHLH; basic Helix-Loop-Helix, PYE; POPYE, BTS; BRUTUS, HHE; histidine, histidine, and glutamic acid-containing domain, HORZ1; Hemerythrin motif-containing protein without RING- and Zn-finger 1, IMA1/2; IRON MAN/ FE uptake inducing Peptide, DTPA; Diethylenetriamine-N,N,N',N'',N''-pentaacetic acid Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and material The datasets supporting the conclusions of this study are included in this article and its additional files. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the following projects raised by the Research Institute of Tokyo University of Agriculture [Strategic Research Project for 2015-2016, Dean-led Research Project for 2018-2020, and Kome (rice) Project for 2021-2025] to A.S. Authors' contributions A.S. performed the initial screening of TCM mutant lines, designed and conducted subsequent experiments, analyzed data, and was the major contributor to manuscript writing. T.Kumamaru. generated the TCM mutants. J.K. and M.S. conducted the secondary screening of candidate lines. K.N. and H.I. made initial crosses between the tetsu ( hrz1 ) mutant and other rice varieties. A.H., M.K., and K.S. developed and selected rice RILs. S.K., S.Nakayama, and N.K. performed yield surveys and elemental analyses. N.K. also cultivated plants and extracted DNA for GRAS-Di analysis. H.N. and A.K. performed the dCAPS analysis. R. Shimokawa and S.M. conducted alkaline soil tests. S.Nishino, T.Kobayashi, K.Y., R.Sugano, and H.H. carried out generation-advancement cultivation and Fe analysis of Fe-biofortified lines. T.O., Y.S., T.Kumamaru, S.U., T.F., and K.H. provided essential guidance and supported experiments, discussions, and manuscript revision. Acknowledgements The rice seeds of O. sativa cv. The T65 (T0504) cells were provided by the National Institute of Genetics (Japan) to A.S. We are grateful to Kayoko Aizawa for the excellent technical assistance during primary screening at the University of Tokyo. We also thank all the members of the research projects at TUA, especially Yumi Aizawa, Tsukasa Suzuki, Hiroyuki Oshima, Hirofumi Inoue, Daiki Oka, Akinobu Kajikawa, Haruko Noguchi, Yoshimasa Tsujii, Hiroko Suzuno, Toshimori Kadokura, Kazuhiro Homma, Yuji Yamamoto, Kazuko Yamaguchi-Shinozaki, and Takuji Sasaki. The authors acknowledge the use of a rice paddy field in the Kanto region of Japan for the cultivation trials, supported by Taku Kato of TUA and the field owners Mr. Mrs. Hideo and Sachiko Noji. References Aoyama T, Kobayashi T, Takahashi M, Nagasaka S, Usuda K, Kakei Y, Nishizawa NK (2009) OsYSL18 is a rice iron (III)–deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints. Plant Mol Biol 70(6):681–692. https://doi.org/10.1007/s11103-009-9500-3 Ariga T, Hazama K, Yanagisawa S, Yoneyama T (2014) Chemical forms of iron in xylem sap from graminaceous and non-graminaceous plants. 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Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Nanami","middleName":"","lastName":"Kawano","suffix":""},{"id":541978483,"identity":"a701b09c-aff6-4a25-8bca-73b85f5e4ec4","order_by":11,"name":"Shunta Nishino","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Shunta","middleName":"","lastName":"Nishino","suffix":""},{"id":541978487,"identity":"ef11a48d-46c3-43e0-b7cc-29638b6e7e17","order_by":12,"name":"Takehiro Kobayashi","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Takehiro","middleName":"","lastName":"Kobayashi","suffix":""},{"id":541978490,"identity":"92f658af-64cc-4b07-92d0-6693ed1c9dae","order_by":13,"name":"Haruka Nakamura","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Haruka","middleName":"","lastName":"Nakamura","suffix":""},{"id":541978491,"identity":"4de756ca-eab8-4f4d-8723-727f4aa5f67e","order_by":14,"name":"Kurumi Yamanaka","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Kurumi","middleName":"","lastName":"Yamanaka","suffix":""},{"id":541978492,"identity":"806fea04-0837-4c82-b626-500062da2700","order_by":15,"name":"Ayane Konno","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Ayane","middleName":"","lastName":"Konno","suffix":""},{"id":541978493,"identity":"571f82d2-5b31-413c-8342-d6dbea98eb39","order_by":16,"name":"Rina Shimokawa","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Rina","middleName":"","lastName":"Shimokawa","suffix":""},{"id":541978495,"identity":"f16d820c-df00-4c63-bd66-f4337d9fddb9","order_by":17,"name":"Ryoma Sugano","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Ryoma","middleName":"","lastName":"Sugano","suffix":""},{"id":541978496,"identity":"768ae893-c54e-4cde-8a9e-e08e54d2d266","order_by":18,"name":"Shuhei Mukaida","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Shuhei","middleName":"","lastName":"Mukaida","suffix":""},{"id":541978497,"identity":"b76f6055-3d1a-4ff3-8f99-c5b7b432cdc2","order_by":19,"name":"Hayate Hata","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Hayate","middleName":"","lastName":"Hata","suffix":""},{"id":541978499,"identity":"a1e0ab78-297a-46d5-b1cb-1d7694c16066","order_by":20,"name":"Takuji Ohyama","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Takuji","middleName":"","lastName":"Ohyama","suffix":""},{"id":541978500,"identity":"c782311f-9bbc-48f0-9531-9194ca28f706","order_by":21,"name":"Yusuke Shikanai","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Yusuke","middleName":"","lastName":"Shikanai","suffix":""},{"id":541978501,"identity":"02a9ca8f-b277-4b41-bef9-f498e541d013","order_by":22,"name":"Toshihiro Kumamaru","email":"","orcid":"","institution":"Kyushu University","correspondingAuthor":false,"prefix":"","firstName":"Toshihiro","middleName":"","lastName":"Kumamaru","suffix":""},{"id":541978503,"identity":"6425e021-80f9-4679-9778-d856c781cd0a","order_by":23,"name":"Shimpei Uraguchi","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Shimpei","middleName":"","lastName":"Uraguchi","suffix":""},{"id":541978504,"identity":"668bb2e6-fa25-46b5-bb49-9b9fe558bdc1","order_by":24,"name":"Toru Fujiwara","email":"","orcid":"","institution":"The University of Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Toru","middleName":"","lastName":"Fujiwara","suffix":""},{"id":541978505,"identity":"100ff7c7-652d-458a-bfe2-8d42bea3995d","order_by":25,"name":"Kyoko Higuchi","email":"","orcid":"","institution":"Tokyo University of Agriculture","correspondingAuthor":false,"prefix":"","firstName":"Kyoko","middleName":"","lastName":"Higuchi","suffix":""}],"badges":[],"createdAt":"2025-10-27 11:40:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7955540/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7955540/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12284-026-00897-6","type":"published","date":"2026-03-13T15:58:15+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":95801402,"identity":"fdc7962d-9033-4505-9f97-52f94ce500fc","added_by":"auto","created_at":"2025-11-13 08:25:19","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165278,"visible":true,"origin":"","legend":"","description":"","filename":"RiceSaitoetal2025v251027.docx","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/e1415e5b8dd59952d2ddbe18.docx"},{"id":95743957,"identity":"7bd42513-7c74-4305-8c97-de8256339e0d","added_by":"auto","created_at":"2025-11-12 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08:25:29","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":297139,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/932d786fb0e13b268ec19ceb.html"},{"id":95743949,"identity":"fd1c1323-2d3b-4e35-9157-7a3bd8377443","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":203532,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003etetsu \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutant accumulates high concentrations of Fe and Mn in the shoot.\u003c/strong\u003e Fe concentrations in shoots (a) and xylem sap (b) of ‘Taichung-65’ (T65) as wild-type and TCM1587 mutant. Plants were grown for 37 days on soil in an air-conditioned greenhouse (24 ± 3°C) under natural light. Each column is the mean ± standard error with significant differences between T65 and \u0026nbsp;TCM1587 determined using a Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003en\u003c/em\u003e = 15 and \u003cem\u003en\u003c/em\u003e = 22 for shoots of WT and TCM1587, respectively, and \u003cem\u003en\u003c/em\u003e = 6 and \u003cem\u003en\u003c/em\u003e = 24 for xylem saps of T65 and TCM1587, respectively). Fe, Mn, Zn, and Cu concentrations in shoots (c, d, e, and f) and roots (g, h, i, and j) of T65 and \u003cem\u003etetsu\u003c/em\u003e mutant selected from TCM1587. Plants were grown for 26 days on soil in the growth chamber at 28°C and a light intensity of 200 μmol photon m\u003csup\u003e−2\u003c/sup\u003e s\u003csup\u003e−1\u003c/sup\u003e\u0026nbsp;under a 14 h light/10 h dark cycle. Each column is the mean ± standard error with significant differences (***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) between T65 and \u003cem\u003etetsu\u003c/em\u003e using Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003en\u003c/em\u003e = 4–5).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/80e6a04f923e3196c35a98d5.png"},{"id":95743951,"identity":"9ee15415-b91b-4655-9b56-92ed5cc46478","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":562102,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003etetsu\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant seeds accumulate high levels of Fe, Zn, and Cu.\u003c/strong\u003e Fe, Mn, Zn, and Cu concentrations in brown (a, b, c, and d) and polished (e, f, g, and h) T65 and \u003cem\u003etetsu\u003c/em\u003e mutant rice. T65 and \u003cem\u003etetsu\u003c/em\u003e mutant plants were grown on soil until fully ripe in a controlled greenhouse under natural light and supplemental light with a light intensity of more than 400 µmol m\u003csup\u003e-2\u003c/sup\u003e\u0026nbsp;s\u003csup\u003e-1\u003c/sup\u003e\u0026nbsp;during the day, with a 14 h light (28°C)/10 h dark (25°C) cycle. Each column is the mean ± standard error with significant differences (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) between T65 and \u003cem\u003etetsu\u003c/em\u003e using Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003en\u003c/em\u003e = 3).\u0026nbsp; The polishing rates of polished T65 and \u003cem\u003etetsu\u003c/em\u003e used for metal measurements did not significantly differ (i). Fe localization images obtained from Perls’ staining on the seed the surface side and inside (j).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/ecc16dde9db10a380745d41a.png"},{"id":95743952,"identity":"bba2941c-948b-4097-ad77-2ad26ae54ffd","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":573396,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003etetsu\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant tolerates Fe-deficient alkaline soil conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSoil pH and concentration of DTPA-extracted forms of Fe (a) in the soils used. Typical images of T65 and \u003cem\u003etetsu\u003c/em\u003e grown in soil at pH 5.0 and 8.7 during the heading period (b). Changes in SPAD values of plants grown in soils with different pH values (c). Young seedlings were pre-cultured in soil (pH 5.0) for 38 days and then transferred to soil at pH 5.0 or 8.7. The SPAD values were measured periodically for 101 days after transplantation. In soil with a pH of 5.0, leaves yellowed after 80 days owing to senescence during ripening. After harvest, the following data on yield and Fe content were collected: dry weight of shoots (d), effective tillering rate (e), image of harvested brown rice (f), and Fe concentrations in straw (g) and brown rice (h). Data represent the mean ± standard error with significant differences between T65 and \u003cem\u003etetsu\u003c/em\u003e using Student’s \u003cem\u003et\u003c/em\u003e-test (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, \u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/8de5243e264df0f7b53eadfa.png"},{"id":95743950,"identity":"ab094bbd-25e0-485e-8b80-42cb471acd86","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":443670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsHRZ1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene emerged as the causative gene for increased Fe content in the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etetsu\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant.\u003c/strong\u003e (a) Schematic diagram of the breeding steps to obtain F\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;lines used for the genetic analysis. (b) Frequency distribution of Fe content in shoots of \u003cem\u003etetsu\u003c/em\u003e mutant, ‘Asamurasaki,’ and their F\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;populations (\u003cem\u003en\u003c/em\u003e = 235). The chi-square test predicted that the causative gene was a single recessive gene. (c) The genomic location of the amplicon markers for distinguishing between genomes of \u003cem\u003etetsu\u003c/em\u003e mutants and ‘Asamurasaki.’ (d) Δ(amplicon index) was developed to visualize the genome position of the causal gene after GRAS-Di analysis for the F\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;population. (e) Schematic diagram of a nonsense mutation found within the \u003cem\u003eOsHRZ1\u003c/em\u003e gene in the candidate region (24.3–30.3 Mb) on chromosome 1 of the \u003cem\u003etetsu\u003c/em\u003e mutant. Details are shown in Table S1. (f) Mutations within the \u003cem\u003eOsHRZ1\u003c/em\u003e gene present in the \u003cem\u003etetsu\u003c/em\u003e mutant reconfirmed using the Sanger sequencing of three independent plants. Arrows indicate the position of the mutated base (C to T).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/ba8b127607f29b71b4a9161d.png"},{"id":95802118,"identity":"7422dbc3-d895-491c-9ed3-ad2453acc852","added_by":"auto","created_at":"2025-11-13 08:26:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":147069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003etetsu\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-type homozygous mutation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsHRZ1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e markedly increases Fe in the shoot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a) Comparison of the primary sequence of the OsHRZ1 protein between WT and \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant based on the DNA sequence. The major functional domains within the OsHRZ1 protein are also shown in the figure. (b) A dCAPS marker was developed for genotyping by detecting the presence or absence of the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation. Details of this dCAPS method are described in Fig. S6. (c) Comparison of shoot Fe concentrations in different genotypes of the F\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;population from crosses between \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) and ‘Asamurasaki.’ Shoots of the 2-week-old F\u003csub\u003e2\u003c/sub\u003e\u0026nbsp;population rice plants grown in soil were harvested and analyzed for Fe. Means with the same letter are not significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, according to Tukey’s multiple comparison test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/1ea1c9b440fd29b49c118bc7.png"},{"id":95743955,"identity":"c60abc96-cbfa-4e6d-aeaa-e1da3c40b0c7","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":975181,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRILs with the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etetsu\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehrz1)\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003emutation show sufficient fertility and well-developed seed formation. \u003c/strong\u003e(a) TCM1587, which is the original ancestral lineage of the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant, exhibited low fertility and small panicles. The typical undeveloped seeds are indicated by the arrow. (b) Schematic diagram of the breeding steps to obtain the RILs from the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) and “Asamurasaki.” (c–d) Image of plants at heading period and the panicle taken from three individual plants. RILs showing the normal appearances. (e) Characteristics of brown rice (anthocyanin coloration and waxy properties). (f) dCAPS analysis showing three RILs have a homozygous \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/a9b3a616107125abb642e691.png"},{"id":95743958,"identity":"74f0b723-a8db-4ebd-b982-42c77c2d5879","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":383981,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRILs with the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003etetsu\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehrz1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e) mutation exhibit high-Fe accumulating characteristics with normal grain yield.\u003c/strong\u003e RILs improved the grain number per panicle (a) and total grain yield (b) compared with those in TCM1587, the original ancestral lineage of the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant. Fe concentrations in shoots (c), brown rice (d), and polished rice (e) were significantly increased in RILs with \u003cem\u003etetsu\u003c/em\u003e (\u003cem\u003ehrz1\u003c/em\u003e) mutation compared with their parental cultivars, T65 and ‘Asamurasaki.’ The total shoots and brown rice harvested after maturity were analyzed for the same rice used in Fig. 6. Plants were grown on soil until fully ripe in a controlled greenhouse under natural light and supplemental light with a light intensity of more than 400 µmol m\u003csup\u003e-2\u003c/sup\u003e\u0026nbsp;s\u003csup\u003e-1\u003c/sup\u003e\u0026nbsp;during the day, with a 14 h light (28°C)/10 h dark (25°C) cycle. Of the RILs, A-5BM accumulated higher concentrations of anthocyanins and phenolic compounds in brown rice than T65 (f–i). Each column represents the mean ± standard error. Means with the same letter are not significantly different at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, according to Tukey’s multiple comparison test or with asterisks indicating significant differences (* \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001) between T65 and \u003cem\u003etetsu\u003c/em\u003e using Student’s \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003en\u003c/em\u003e = 3).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/20483d86a2b7cd61c8457a30.png"},{"id":104739441,"identity":"bb084623-d9bb-4a71-9cbe-b14ea455ca06","added_by":"auto","created_at":"2026-03-16 16:06:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5399085,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/4f39f993-efcf-40a4-a94a-0d05462b8b48.pdf"},{"id":95743956,"identity":"006ddc71-6dbd-4264-b077-91da104f3ab3","added_by":"auto","created_at":"2025-11-12 14:21:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1174196,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigsSaitoetal.2025tetsuFebiofortifiedricev20251023.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7955540/v1/602dbaebe767df67142569b3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment of a non-transgenic iron-biofortified rice line using a novel HRZ1 mutation","fulltext":[{"header":"Background","content":"\u003cp\u003eFe is an essential element for most living organisms, coordinating with many enzymes responsible for electron transfer and redox reactions to produce energy, mainly through photosynthesis and respiration (Kermeur et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Fe deficiencies are among the most prevalent micronutrient deficiencies worldwide, affecting 2\u0026nbsp;billion people and causing more than 0.8\u0026nbsp;million deaths annually (Murgia et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; WHO 2019). Biofortification is a cost-effective and sustainable approach that aims to solve this problem by boosting the micronutrient content of crops (Connorton and Balk \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Rice (\u003cem\u003eOryza sativa\u003c/em\u003e) is a staple food for over 3.5\u0026nbsp;billion people and comprises approximately 23% of the calories consumed worldwide (Khush \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hackl et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kermeur et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The development of Fe biofortified rice is a solution to the global problem of Fe deficiency anemia (Hackl et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eRice plants acquire Fe from soil as ferrous Fe (Fe\u003csup\u003e2+\u003c/sup\u003e) through OsIRT1, OsIRT2, and OsNRAMP1 (Ishimaru et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), and as the phytosiderophore 2\u0026prime;-deoxymugineic acid (DMA)\u0026ndash;Fe\u003csup\u003e3+\u003c/sup\u003e complex (Takagi \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Curie et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) through Yellow Stripe 1 (YS1)/YSL transporters, such as OsYSL15 (Curie et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2001\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Inoue et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lee and An 2009; Suzuki et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). DMA synthesis in rice is catalyzed by nicotianamine synthase (NAS) to produce the Fe\u003csup\u003e2+\u003c/sup\u003e chelator nicotianamine (NA) from three molecules of methionine (Higuchi et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), followed by the conversion of NA to DMA via nicotianamine aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS) (Takahashi et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1999\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Inoue et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Bashir et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A transporter of mugineic acid family phytosiderophores (TOM1) is responsible for transporting DMA from the roots into the rhizosphere (Nozoye et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Almost all the genes involved in above Fe uptake mechanism are strongly upregulated in response to Fe deficiency (Kobayashi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAfter Fe absorption, OsYSL2 and other YSLs support the long-distance translocation of Fe as MA\u0026ndash;Fe\u003csup\u003e3+\u003c/sup\u003e and NA\u0026ndash;Fe\u003csup\u003e2+\u003c/sup\u003e complexes from the roots to shoots (Ishimaru et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Koike et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Aoyama et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Senoura et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, FERRIC REDUCTASE DEFECTIVE LIKE 1 (OsFRDL1) is a transporter involved in the efflux of citrate into the xylem for efficient Fe translocation (Ariga et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Yokosho et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Other transporters are also involved in Fe transport/translocation: OsPEZ1/2, which facilitates Fe transport by effluxing phenolic compounds with Fe-chelating activity (Bashir et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e; Ishimaru et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011a\u003c/span\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003eb\u003c/span\u003e), EFFLUX TRANSPORTER OF NA 1/2 (OsENA1/2); and OsTOMs NA efflux transporters (Nozoye et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Fe transported into the cell is further distributed to organelles by the vacuolar Fe transporter OsVIT1/2 (Zhang et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Che et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and the mitochondrial Fe transporter MIT1 (Bashir et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2011b\u003c/span\u003e). The vacuolar MA transporter, OsVMT, also plays a key role in controlling the subcellular partitioning of MAs, thereby regulating metal translocation to grains (Che et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Excess Fe within cells is sequestered in the vacuoles and the Fe-storage protein ferritin (OsFER1 and OsFER2) (Stein et al. \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Briat et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAttempts have been made to generate Fe-enhanced rice plants by controlling the expression of individual genes or a combination of genes responsible for the transport, translocation, and accumulation of Fe using genetic engineering techniques (Masuda et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kawakami and Bhullar \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition to these Fe transport- and storage-related genes, manipulation of the transcriptional and post-translational regulators of Fe accumulation could potentially be utilized for Fe biofortification. For example, overexpression of the transcription factors IDEF1 and IRON-REGULATED TRANSCRIPTION FACTOR 2 (OsIRO2) or IRON-REGULATED TRANSCRIPTION FACTOR 3 (OsIRO3) knockout enhances Fe-deficiency responses and increases Fe acquisition. Here, the transcription factors IDEF1 and IDEF2, located upstream of the Fe signaling pathway in rice, positively regulate the early Fe-deficiency response, primarily for most genes involved in DMA\u0026ndash;Fe (III) and Fe\u003csup\u003e2+\u003c/sup\u003e uptake (Kobayashi et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ogo et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The basic helix-loop-helix (bHLH) transcription factor OsIRO2 positively regulates Fe deficiency\u0026ndash;induced gene expression under the control of IDEF1. The bHLH gene OsIRO3, a homolog of POPYE (PYE) in Arabidopsis, negatively regulates Fe deficiency\u0026ndash;inducible genes (Ogo et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Zheng et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAs post-translational regulators, the hemerythrin motif\u0026ndash;containing RING zinc-finger proteins (HRZ1 and HRZ2), which function as E3-ubiquitin ligases and homologs of BRUTUS (BTS) in Arabidopsis, negatively control most known Fe deficiency\u0026ndash;inducible genes, including the bHLH transcription factors OsIRO2 and OsIRO3, by degradation via the 26S proteasome. Notably, HRZs contain histidine, histidine, and glutamic acid (HHE) domains with direct Fe- and Zn-binding properties and are thus considered promising candidates for Fe sensors in rice cells (Kobayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pullin et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). HRZs also negatively regulate the expression of OsIMA1 and OsIMA2, which are rice orthologs of the small signaling peptide IRON MAN (IMA)/Fe UPTAKE INDUCING PEPTIDE (FEP) found in Arabidopsis (Grillet et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), at the transcriptional level via an unknown pathway (Kobayashi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eHRZ2\u003c/em\u003e knockdown reportedly enhances Fe deficiency tolerance and Fe accumulation (Kobayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Regarding HRZ1, detailed phenotypic analysis of the previous \u003cem\u003ehrz1\u003c/em\u003e mutant had not been advanced due to its poor growth phenotype. Recently, several transformants and genome-edited rice plants that accumulate the mutated HRZ1 protein lacking the C-terminal region have been created using a transgenic strategy and a genome-editing technique, and exhibited enhanced Fe-deficiency responses, similar to the suppression of \u003cem\u003eHRZ2\u003c/em\u003e (Shinkawa et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, since all of these have undergone genetic modification, their yield potential and practicality for use as Fe-biofortified rice have not been examined.\u003c/p\u003e\u003cp\u003eIn this study, we developed practical Fe-biofortified rice lines by isolating a rice mutant with high Fe content in the shoot from a screening of 3,000 MNU-mutagenized mutant lines. We identified a novel nonsense mutation in the fourth exon of the \u003cem\u003eHRZ1\u003c/em\u003e gene, which corresponds to the beginning of the second hemerythrin domain of HRZ1 proteins. This mutation exhibited a pronounced phenotype characterized by Fe accumulation throughout the shoot, including in leaves and grains. This is the first report to demonstrate that the discovered \u003cem\u003eHRZ1\u003c/em\u003e mutation is beneficial for producing non-transgenic, practical, biofortified rice plants.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIsolation of a rice mutant\u003c/b\u003e \u003cb\u003etetsu\u003c/b\u003e \u003cb\u003ewith high Fe content in shoots and xylem sap\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA rice mutant that accumulated high amounts of Fe was screened from an MNU-mutagenized T65 mutant population (TCM lines). As part of the primary screening, we performed an ionome analysis of the shoots and xylem sap of 21-day-old seedlings of the mutant population, as previously reported (Tanaka et al. \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Of the 2,704 germinated TCM lines, one mutant line, TCM1587, had higher Fe concentrations in both the shoot and xylem sap (9- and 6.5-fold on average, respectively) than wild-type T65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). As a secondary screening step, prompted by the large variation in Fe content within TCM1587, we selected one progeny line with a fixed high Fe accumulation trait, showing 3- and 4-fold increases in shoot Fe and Mn, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Zn and Cu concentrations in shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f) and Fe, Mn, Zn, and Cu concentrations in roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg\u0026ndash;j) were not significantly different between T65 and the progeny line. Hereafter, we refer to this line as \u003cem\u003eTransporting Errors in TranSition metal Uptake (tetsu)\u003c/em\u003e: \u003cem\u003etetsu\u003c/em\u003e means iron in Japanese.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003etetsu\u003c/b\u003e \u003cb\u003emutant does not absorb excess toxic heavy metals\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBecause the \u003cem\u003etetsu\u003c/em\u003e mutant accumulated higher concentrations of Fe and Mn than did T65, we assumed an increase in the accumulation of other heavy metals such as Ni, Co, Cd, and Pb. To clarify this, the germinated seeds of this mutant were grown in soil containing low concentrations of Ni, Co, Cd, and Pb (10 mg kg soil \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Ni and 1 mg kg soil \u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for Co, Cd, and Pb), and the amount of heavy metal absorption was measured (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea, b). The results showed a 1.8-fold increase in the essential element Ni in the shoots and roots of the \u003cem\u003etetsu\u003c/em\u003e mutant compared with that in T65, which was mostly stored in the roots and minimally transferred to the shoots (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ec, d). Other non-essential heavy metals (Co, Cd, and Pb) accumulated equally in the \u003cem\u003etetsu\u003c/em\u003e mutant and T65, both in the roots and shoots (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ee\u0026ndash;g), confirming that \u003cem\u003etetsu\u003c/em\u003e did not excessively accumulate these heavy metals.\u003c/p\u003e\u003cp\u003e\u003cb\u003etetsu\u003c/b\u003e \u003cb\u003emutant accumulates Fe in grains\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, we measured the metal content of brown and polished rice. In the brown rice of the \u003cem\u003etetsu\u003c/em\u003e mutant, Fe increased more than 2-fold, Zn and Cu also increased significantly by over 40%, whereas Mn decreased significantly compared with that in T65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;d). In polished rice, the \u003cem\u003etetsu\u003c/em\u003e mutant exhibited approximately 2-fold higher Fe, 1.3-fold higher Zn, and 1.5-fold higher Cu than those in T65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u0026ndash;h). Because we prepared polished rice at the same milling ratio of 90% between T65 and \u003cem\u003etetsu\u003c/em\u003e, the amount of remaining bran did not affect this difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). Thus, we concluded that \u003cem\u003etetsu\u003c/em\u003e had significantly higher levels of Fe and moderately higher levels of Zn and Cu in the endosperm than did T65. To further verify the high Fe accumulation traits in grains, we analyzed Fe localization in rice grains using Perls\u0026rsquo; staining. The stained images visually revealed that Fe was not limited to the outer layers (pericarp and aleurone layer) and scutellum, but also accumulated in the endosperm of the \u003cem\u003etetsu\u003c/em\u003e mutant compared with that in T65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003etetsu\u003c/b\u003e \u003cb\u003emutant has significant alkaline soil tolerance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHigh Fe accumulation in the shoots of the \u003cem\u003etetsu\u003c/em\u003e mutant was also expected to contribute to Fe deficiency tolerance. To confirm this possibility, Fe-deficiency tolerance was investigated in a calcareous alkaline soil environment using culture soil with calcium hydroxide [Ca(OH)\u003csub\u003e2\u003c/sub\u003e]. At a concentration of 1.5% (w/w) Ca(OH)\u003csub\u003e2\u003c/sub\u003e in the rice culture soil (pH 5.0), soil pH increased to 8.7, and the amount of available Fe decreased to approximately 30% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and Fig. S2), which is typical for calcareous Fe-deficient soils. The high pH persisted throughout cultivation; therefore, Ca(OH)\u003csub\u003e2\u003c/sub\u003e was only mixed before planting.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA high Fe-containing F\u003csub\u003e3\u003c/sub\u003e population backcrossed with the \u003cem\u003etetsu\u003c/em\u003e mutant to T65 was used to exclude the effects of mutations not involved in Fe accumulation in the \u003cem\u003etetsu\u003c/em\u003e mutant. After 38 days of pre-cultivation in pH 5.0 control soil, plants were transplanted into pH 5.0 or 8.7 soil for an additional 101 days. At pH 5.0, the appearance (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), SPAD value of the latest expanded leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), shoot weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), and effective tillering rate (percentage of tillers that led to panicle formation) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee) were similar between WT and \u003cem\u003etetsu\u003c/em\u003e homozygous plants with no significant difference. Conversely, at pH 8.7, the WT showed significantly lower SPAD values in young leaves than did \u003cem\u003etetsu\u003c/em\u003e at 37 days after transplanting, and although transient recovery was observed, the SPAD values of WT continued to decrease until the end of cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Meanwhile, \u003cem\u003etetsu\u003c/em\u003e maintained young leaf color at the same level as that at pH 5.0 during the cultivation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Growth at pH 8.7 was equally reduced in both WT and \u003cem\u003etetsu\u003c/em\u003e compared with that at pH 5.0 under alkaline conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, d), but the effective tillering rate was significantly higher than that in WT at pH 8.7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). At the end of cultivation (101 days after transplanting), seeds in WT were still green and immature, whereas many of the seeds in the \u003cem\u003etetsu\u003c/em\u003e mutant had almost reached full maturity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). The Fe concentration in the shoots and brown rice was significantly higher in the \u003cem\u003etetsu\u003c/em\u003e mutant than that in WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, h).\u003c/p\u003e\u003cp\u003eSince both the WT and \u003cem\u003etetsu\u003c/em\u003e mutant plants had few seeds at pH 8.7, accurate analysis of yield components was not possible; therefore, we conducted a second trial to confirm the above results under milder alkaline conditions using 1.0% Ca(OH)\u003csub\u003e2\u003c/sub\u003e, where the soil pH was approximately 8.0, with the DTPA-Fe level significantly unchanged at the beginning of cultivation compared with that at pH 5.0 (Fig. S2). As shown in Fig. S3a, the grain weight of the \u003cem\u003etetsu\u003c/em\u003e mutant did not change between pH 5.0 and 8.0, whereas that of the WT significantly decreased to approximately 60% at pH 8.0. Total straw weight and effective tillering rate remained unchanged (pH 8.0) in the \u003cem\u003etetsu\u003c/em\u003e mutant, but were decreased in WT at pH 8.0 (Fig. S3b, c). The number of panicles showed no significant difference; however, that in WT tended to decrease at high pH, whereas the \u003cem\u003etetsu\u003c/em\u003e mutant showed no change (Fig. S3d). WT and the \u003cem\u003etetsu\u003c/em\u003e mutant did not significantly differ in the number of tillers, grain weight per panicle, and grain-filling rate (Fig. S3d\u0026ndash;h).\u003c/p\u003e\u003cp\u003eTaken together, we confirmed that the \u003cem\u003etetsu\u003c/em\u003e mutant was tolerant to alkaline Fe-deficient conditions, maintaining source size (total straw weight) and effective tillering rate, resulting in higher yield in the \u003cem\u003etetsu\u003c/em\u003e mutant than in the WT under these conditions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHigh Fe accumulation trait in\u003c/b\u003e \u003cb\u003etetsu\u003c/b\u003e \u003cb\u003eis associated with a novel nonsense mutation in\u003c/b\u003e \u003cb\u003eHRZ1\u003c/b\u003e\u003c/p\u003e\u003cp\u003eNext, to identify the gene responsible for the high Fe accumulation of \u003cem\u003etetsu\u003c/em\u003e, we crossed the the \u003cem\u003etetsu\u003c/em\u003e mutant with the Japanese glutinous black\u0026ndash;purple rice cultivar \u0026lsquo;Asamurasaki\u0026rsquo; with normal Fe and high functional compounds in grains (Pereira-Caro et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Oo et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) to obtain an F\u003csub\u003e2\u003c/sub\u003e population in which phenotypic segregation could be easily identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). A total of 219 F\u003csub\u003e2\u003c/sub\u003e plants were grown in soil for 16\u0026ndash;18 days after germination, and the Fe concentration of the whole shoot was measured. A total of 166 F\u003csub\u003e2\u003c/sub\u003e individuals had Fe concentrations similar to those of \u0026lsquo;Asamurasaki,\u0026rsquo; while 53 F\u003csub\u003e2\u003c/sub\u003e individuals had Fe concentrations as high as those of the \u003cem\u003etetsu\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The chi-square test did not reject the null hypothesis of a 3:1 segregation ratio, which is consistent with the idea that the causal gene was single and recessive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo identify the causal gene, the genotypes of F\u003csub\u003e2\u003c/sub\u003e individuals were determined using a recently developed GRAS-Di system (Hosoya et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Miki et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fekih et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), a method based on comprehensive amplicon sequencing. First, a comparison of the amplicons of the parents (\u003cem\u003etetsu\u003c/em\u003e and \u0026lsquo;Asamurasaki\u0026rsquo;) revealed 2,090 amplicon markers. These DNA markers mapped widely and uniformly to all rice chromosomes, confirming that a set of good-quality markers had been obtained to identify the causal gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec and Fig. S4). Next, we developed an \u0026ldquo;amplicon index\u0026rdquo; that quantified the detection or non-detection of each marker in individual F\u003csub\u003e2\u003c/sub\u003e plants. The Δ (amplicon index) was calculated to identify regions where marker detection rates differed significantly between F\u003csub\u003e2\u003c/sub\u003e populations with low and high Fe (Fig. S5).\u003c/p\u003e\u003cp\u003eThis method successfully narrowed down the causative locus to the 24.3- to 30.3-Mb region of chromosome 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). To identify all the mutation sites of \u003cem\u003etetsu\u003c/em\u003e within this causative locus, we compared the whole genome sequences of T65 and \u003cem\u003etetsu\u003c/em\u003e using high-precision next-generation sequencing analysis and identified 39 mutation sites. These were all GC/AT substitutions, a typical type of mutation known to occur after MNU mutagenesis. Among these, we found a novel nonsense mutation within the fourth exon of the \u003cem\u003eHRZ1\u003c/em\u003e gene, which encodes a ubiquitin ligase predicted to function as a negative regulator of Fe-uptake/translocation-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f). Other mutation sites included missense mutations in non-coding and untranslated regions, as well as putative genes with no significant reported function, none of which were potential candidates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eShoot Fe content is increased by the\u003c/b\u003e \u003cb\u003etetsu\u003c/b\u003e\u003cb\u003e-type\u003c/b\u003e \u003cb\u003eHRZ1\u003c/b\u003e \u003cb\u003emutation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe mutation found in \u003cem\u003eHRZ1\u003c/em\u003e is presumed to express a truncated HRZ1 protein that has only the first HHE domain without the other two HHE domains and the C-terminal zinc-finger domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The protein that can be produced by such a mutation is similar to the rice hemerythrin domain protein HORZ1(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), which positively regulates the Fe-deficiency response\u0026mdash;the opposite function to the normal HRZs (Kobayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shinkawa et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe developed a dCAPS marker that could efficiently detect this genetic mutation using modified forward primers and the HincII enzyme (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and Fig. S6). This dCAPS assay reliably detected the \u003cem\u003eHRZ1\u003c/em\u003e gene as a 309 bp fragment in WT, a 261 bp fragment in the homozygous mutant, and both fragments in the heterozygous mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Investigating the relationship between \u003cem\u003eHRZ1\u003c/em\u003e genotype and shoot Fe concentration in 114 F\u003csub\u003e2\u003c/sub\u003e plants from \u003cem\u003etetsu\u003c/em\u003e\u0026times;\u0026lsquo;Asamurasaki\u0026rsquo; discriminated by this dCAPS marker, we found that the homozygous mutant of \u003cem\u003eHRZ1\u003c/em\u003e had significantly higher Fe content than did WT or heterozygous plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). This result suggests that the high Fe accumulation in the \u003cem\u003etetsu\u003c/em\u003e mutant was caused by a \u003cem\u003etetsu\u003c/em\u003e-type nonsense mutation in \u003cem\u003eHRZ1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003etetsu(hrz1)\u003c/b\u003e \u003cb\u003emutant strongly induces an Fe-deficiency response at the transcriptional level\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePreviously, RNAi-knockdown lines of \u003cem\u003eHRZ2\u003c/em\u003e showed increased expression of various Fe uptake- and utilization-related genes in Fe-sufficient roots, similar to Fe-deficient non-transformant rice roots (Kobayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). To confirm whether the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant also upregulated the expression of such genes, we performed RNA-seq analysis in WT and the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant grown in normal Fe-sufficient soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Because high Fe accumulation occurred in the shoots of \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), we investigated its expression in fully developed young leaves.\u003c/p\u003e\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, a significant increase in the expression of various Fe deficiency\u0026ndash;induced genes was found in the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant compared with that in WT, including \u003cem\u003eIMA1\u003c/em\u003e, \u003cem\u003eIMA2\u003c/em\u003e, \u003cem\u003eIRO2, IRO3\u003c/em\u003e, \u003cem\u003eNAS1, NAS2, YSL2\u003c/em\u003e, \u003cem\u003eNRAMP1\u003c/em\u003e, \u003cem\u003eTOM1\u003c/em\u003e, \u003cem\u003eOPT7\u003c/em\u003e, and \u003cem\u003ePTR\u003c/em\u003e (a putative NA/DMA efflux transporter). HHE domain\u0026ndash;containing proteins (\u003cem\u003eHRZs\u003c/em\u003e and \u003cem\u003eHORZ1\u003c/em\u003e) and IDEF1, which control Fe deficiency signals further upstream, were not significantly altered in the mutant. A significant, but very slight, decrease in IDEF2 expression was observed in the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant. \u003cem\u003eNAS3\u003c/em\u003e levels declined, consistent with previous reports that \u003cem\u003eNAS3\u003c/em\u003e was repressed by Fe deficiency but induced by excess Fe (Aung et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, the expression of the ferritin genes \u003cem\u003eFER1\u003c/em\u003e and \u003cem\u003eFER2\u003c/em\u003e, which are known to be induced by excess Fe, was downregulated in the leaves of \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e). These results clearly indicate that the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant enhanced the Fe-deficiency responses but suppressed the excess Fe responses in the shoot despite containing elevated Fe concentrations. No upregulation of any of the genes encoding proteins involved in the transport and regulation of other heavy metals or metal-chelating compounds (phenolics and citrate) was observed in the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRNA-seq analysis of genes involved in Fe-deficiency responses, DMA synthesis/secretion, Fe transport/translocation, and Fe sequestration/storage in leaves of the wild-type and \u003cem\u003etetsu\u003c/em\u003e mutant\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTranscript ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003e\u003cem\u003etetsu\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(\u003cem\u003ehrz1\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eFold change*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e\u003cp\u003etranscripts per million (TPM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003eHemerythrin domain-containing genes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0689451-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHRZ1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHemerythrin motif-containing RING-\u0026amp; Zinc-finger protein 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.787\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.771\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs05t0551000-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHRZ2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHemerythrin motif-containing RING-\u0026amp; Zinc-finger protein 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.66\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.33\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0861700-00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHORZ1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHemerythrin motif-containing protein without RING- and Zn-finger 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.402\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.175\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.435\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eConstitutively expressed Fe-related transcription factors\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs08t0101000-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIDEF1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIron deficiency-responsive cis-acting element binding factor 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e13.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e11.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.851\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs05t0426200-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIDEF2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIron deficiency-responsive cis-acting element binding factor 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.40\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e49.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.38\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.845\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFe deficiency-induced Fe-related transcriptional and signaling regulators\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0647200-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIMA1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFe-deficiency-inducible IRON MAN 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e613\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e115\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e189\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0061\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0647200-04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIMA1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e12.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2170\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e525\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e173\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0142100-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIMA2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFe-deficiency-inducible IRON MAN 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0967\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2340\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e725\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;10,000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0952800-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIRO2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIron-related transcription factor 2, bHLH056\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00433\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e21.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;10,000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0019\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0379300-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIRO3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eIron-related transcription factor 3, bHLH063\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e22.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e6.3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0004\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e***\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0379300-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIRO3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.84\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e11.9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0044\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0379300-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIRO3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e10.4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eBiosynthesis of mugineic acid family phytosiderophores\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0307300-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNAS1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNicotianamine synthase 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0118\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e177\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0307200-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNAS2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNicotianamine synthase 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.114\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e38.9\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0074\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e**\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0689600-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNAS3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNicotianamine synthase 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.721\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.367\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.048\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e*\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs02t0306401-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNAAT1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNicotianamine amino-transferase 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.39\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.439\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.071\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0237100-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eDMAS1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDeoxymugineic acid synthase 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e24.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.390\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.184\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003e\u003cb\u003eFe uptake and/or translocation\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs02t0649900-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eYSL2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFe- and Mn-nicotianamine transporter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0424\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e4.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e519\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0058\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0258400-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNRAMP1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHomologues of mammalian Nramp1, Fe transporter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.0514\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.05\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e36.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e6.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e718\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0258400-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNRAMP1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;10,000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0036\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0258400-03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNRAMP1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.088\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs11t0134900-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eTOM1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEfflux transporter of mugineic acids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.528\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e\u0026gt;\u0026thinsp;10,000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e***\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0751100-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eOPT7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eOligopeptide Transporter 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e1.75\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.013\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0751100-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eOPT7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e2.09\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.0084\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0871500-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePTR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePeptide transporter (Arabidopsis NAET-like)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e44.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e226\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e25.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e5.11\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.002\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e**\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0667500-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIRT1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIron-regulated transporter 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.947\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0667300-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eIRT2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eIron-regulated transporter 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.0375\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs11t0151500-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eENA1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEfflux of nicotianamine 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00498\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.343\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e68.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs06t0695800-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eENA2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eEfflux of nicotianamine 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.630\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.906\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs12t0282000-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eMIR\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMitochondrial iron-regulated gene\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.288\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u0026gt;\u0026thinsp;10,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs04t0463400-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eVIT1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVacuolar Iron Transporter 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e18.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs09t0396900-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eVIT2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVacuolar Iron Transporter 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.783\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.122\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.056\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs11t0106700-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFER1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFerritin 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.71\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.584\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs11t0106700-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFER1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e27.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e131\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e19.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.536\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.029\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs12t0106000-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFER2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFerritin 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e296\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e39.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e151\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e21.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.512\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.032\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003e* Fold-change indicates the expression ratio of \u003cem\u003etetsu\u003c/em\u003e/WT. Bold numbers indicate genes in the \u003cem\u003etetsu\u003c/em\u003e mutant that were significantly upregulated, whereas underlined numbers indicate genes that were significantly downregulated. WT and \u003cem\u003etetsu\u003c/em\u003e homozygotes were isolated from the F\u003csub\u003e2\u003c/sub\u003e population obtained after a single backcross to T65 for the \u003cem\u003etetsu\u003c/em\u003e mutant, with three biological replicates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). Plants were grown under normal soil conditions for 60 days at 26\u0026deg;C in a greenhouse. Young leaves (6th leaf) were harvested at 2:30 p.m. Data are expressed as transcripts per million. Significant differences (* \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) between the WT and \u003cem\u003etetsu\u003c/em\u003e mutants were tested using the Student's t-test.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eRNA-seq analysis of transporters related to heavy metals in leaves of the wild-type and \u003cem\u003etetsu\u003c/em\u003e mutant\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"12\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTranscript ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDescription\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eWT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003e\u003cem\u003etetsu\u003c/em\u003e\u003c/p\u003e\u003cp\u003e(\u003cem\u003ehrz1\u003c/em\u003e)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eFold change*\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e\u003cp\u003e\u003cem\u003ep\u003c/em\u003e-value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c9\" namest=\"c4\"\u003e\u003cp\u003etranscripts per million (TPM)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"12\" nameend=\"c12\" namest=\"c1\"\u003e\u003cp\u003eOther transporters related to translocation of Fe or other metals\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0232800-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eZIP8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZinc transporter 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e96.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e14.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e32.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e10.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.340\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0232800-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eZIP8\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eZinc transporter 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e0.429\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.043\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e*\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0571900-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePEZ1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhenolics efflux zero 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.46\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.686\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0572900-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003ePEZ2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhenolics efflux zero 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e34\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.888\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs12t0133100-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eVMT\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eVacuolar mugineic acid transporter (ZIFL12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.986\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.937\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs03t0216700-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFRDL1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCitrate transporter (Fe-citrate transport)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.29\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.927\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.870\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs10t0206800-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFRDL2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCitrate transporter (Al-induced)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e14.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.88\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.731\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.128\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs10t0206800-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFRDL2\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCitrate transporter (Al-induced)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.759\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs02t0833100-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFRDL3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCitrate transporter, putative\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.09\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.185\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs01t0919100-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFRDL4\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCitrate transporter (Al-induced)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs02t0650300-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eYSL15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFe- and Mn-nicotianamine transporter\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e\u003cp\u003eN.D.\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs06t0560000-02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eFPN1\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFerroportin, intracellular Ni and Co transporter (IREG1)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.55\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.99\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.424\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0257200-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eNRAMP5\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLow cadmium accumulation 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.580\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.613\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.940\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOs07t0232900-01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eHMA3\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHeavy metal ATPase 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.801\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.973\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u0026plusmn;\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.640\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"12\"\u003e* Fold-change indicates the expression ratio of \u003cem\u003etetsu\u003c/em\u003e/WT. Underlined numbers indicate genes that were significantly downregulated in the \u003cem\u003etetsu\u003c/em\u003e mutant. The expression values were obtained from the same dataset, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFirst practical attempt to increase Fe content in rice grain while simultaneously increasing antioxidant components\u003c/h2\u003e\u003cp\u003eSterility occasionally occurred in the original TCM1587 mutant line used for selecting the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant, independent of high Fe accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). This is due to the vast number of genomic mutations that occur randomly in the MNU mutagen of the original \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) genome. Therefore, we further screened lines with improved defective traits from the progeny of the F\u003csub\u003e2\u003c/sub\u003e population (\u003cem\u003etetsu\u003c/em\u003e \u0026times; \u0026lsquo;Asamurasaki\u0026rsquo;).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAfter 10 generations of repeated selfing, we obtained pure recombinant inbred lines (RILs) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). Among the RILs, A-5BM, C-1M, and A-3U showed normal growth and fertility comparable to those of T65 and \u0026lsquo;Asamurasaki\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec,d). Because the parent cultivar \u0026lsquo;Asamurasaki\u0026rsquo; is a glutinous (waxy) black\u0026ndash;purple rice that accumulates high concentrations of many functional components and antioxidants in its grains (Pereira-Caro et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), the selected RILs inherited these traits in various combinations, i.e., A-5BM, which is black\u0026ndash;purple rice and glutinous (waxy), C-1M, which is white rice and glutinous (waxy), and A-3U, which is white rice that is not glutinous (non-waxy) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee). Using the dCAPS assay, we confirmed that the three RILs harbored a \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ef).\u003c/p\u003e\u003cp\u003eCompared with WT T65 and \u0026lsquo;Asamurasaki,\u0026rsquo; the number of grains per spikelet when grown in the normal soil was significantly lower for the original \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant, but comparable for the three RILs (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The total grain weight was also significantly improved in the three RILs compared with that in the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Shoot Fe concentrations in the three RILs remained significantly increased by approximately 3-fold compared with those in WT cultivars (T65 and \u0026lsquo;Asamurasaki\u0026rsquo;) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Fe concentrations in brown rice were also up to 2-fold higher in these RILs than in WT cultivars (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). Fe concentrations in polished rice also showed an increasing trend, averaging 3- to 5-fold higher compared with those in \u0026lsquo;Asamurasaki\u0026rsquo; and 1.6- to 2.7-fold higher than those in T65 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee). Thus, RILs that regained normal reproductive growth through crossing with \u0026lsquo;Asamurasaki\u0026rsquo; maintained stable, high Fe contents across generations, demonstrating that the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation itself is not a harmful mutation that reduces yield potential. Among these RILs, A-5BM had significantly more antioxidant components, such as anthocyanins and various phenolic acids, which are abundant in \u0026lsquo;Asamurasaki\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ef\u0026ndash;i). These results indicate that the identified novel \u003cem\u003eHRZ1\u003c/em\u003e mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars with various beneficial traits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe identified a new nonsense mutation in \u003cem\u003eHRZ1\u003c/em\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) in the Fe-accumulating mutant \u003cem\u003etetsu\u003c/em\u003e. Notably, RILs with the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutation (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) or the backcross line of the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant (Fig. S3) did not show abnormalities in growth or fertility, with an extremely high Fe content in both the leaves and seeds. This contrasts with the previously reported \u003cem\u003ehrz1-1\u003c/em\u003e mutant (Kobayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) or \u003cem\u003ehrz1-2\u003c/em\u003e genome editing line (Zhang et al. \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), both of which are reportedly sterile and/or have poor growth. Because of these undesirable characteristics, achieving Fe biofortification using the previously reported \u003cem\u003eHRZ\u003c/em\u003e knockout/knockdown lines has been challenging. More recently, Shinkawa et al. (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) created rice plants with mutated \u003cem\u003eHRZ1\u003c/em\u003e using a transgenic strategy and CRISPR/Cas9 genome editing techniques. These transformants/mutants targeting \u003cem\u003eHRZ1\u003c/em\u003e exhibited Fe deficiency tolerance and increased Fe content in brown rice, similar to the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant. However, the shoot Fe content, yield potential, and sterility of these transformants/mutants have not been reported in detail.\u003c/p\u003e\u003cp\u003eAmong the \u003cem\u003ehrz1\u003c/em\u003e mutants reported to date, \u003cem\u003ehrz1-1\u003c/em\u003e showed little or no change in shoot Fe compared with that in WT (Kobayashi et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This contradicts the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant, which accumulated more than 3-fold the amount of Fe in the leaves than in the shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The factor that caused differences between the previously reported \u003cem\u003ehrz1\u003c/em\u003e mutants and the current \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant is likely the difference in the mutation site. A previous \u003cem\u003ehrz1-1\u003c/em\u003e had a DNA insertion upstream of the 5'-UTR, causing HRZ1 expression to be moderately suppressed. In contrast, the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant has a nonsense mutation caused by a single-base substitution at the beginning of the second HHE domain, which translates into a truncated HRZ1 with only one complete HHE domain. The shortened structure of the HRZ1 protein was similar to that of another HHE-containing protein, HORZ1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), which functions as a positive regulator of Fe-deficiency responses. Thus, one possibility is that mutated HRZ1, which may accumulate in the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant, could function as a positive regulator of Fe-deficiency responses in this mutant. This hypothesis is supported by the findings of Shinkawa et al. (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which suggest that overexpression of GFP-HRZ1ΔRZ constructs, comprising only two hemerythrin domains of HRZ1 without the other domains, enhances Fe deficiency responses in a dominant-negative manner due to the mutated shortened HRZ1 proteins. Further research is required to confirm our hypothesis regarding the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation.\u003c/p\u003e\u003cp\u003eAmong the genes upregulated in the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), \u003cem\u003eIMA1\u003c/em\u003e, \u003cem\u003eIMA\u003c/em\u003e2, \u003cem\u003eIRO2\u003c/em\u003e, and \u003cem\u003eIRO3\u003c/em\u003e were significantly upregulated at the transcriptional level in shoots. In addition, Fe deficiency\u0026ndash;inducible genes such as \u003cem\u003eYSL2\u003c/em\u003e, \u003cem\u003eTOM1\u003c/em\u003e, \u003cem\u003eENA1\u003c/em\u003e, \u003cem\u003eNRAMP1\u003c/em\u003e, and \u003cem\u003eNAS1/2\u003c/em\u003e were strongly upregulated in the shoots of the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant. Earlier reports have shown that suppressing \u003cem\u003eHRZ1/2\u003c/em\u003e expression using RNAi causes high expression of these Fe-deficiency response genes (Kobayashi et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Thus, the ability of the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant to accumulate high levels of Fe in the shoots is assumed to be the reason for the enhanced Fe-deficiency responses. Furthermore, the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant exhibited a 5-fold increase in the putative NA/DMA efflux transporter \u003cem\u003ePTR\u003c/em\u003e (Os01g0871500) (Nozoye et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and a 2-fold increase in the expression of \u003cem\u003eOPT7\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The gene with high homology to \u003cem\u003ePTR\u003c/em\u003e is \u003cem\u003eNAET\u003c/em\u003e1/2 in Arabidopsis, which is responsible for Fe source-to-sink transport (Chao et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). OPT7 is a transporter responsible for the xylem unloading of Fe\u003csup\u003e2+\u003c/sup\u003e and the preferential distribution of Fe in developing tissues (Bashir et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Yamaji et al. \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, the increased expression of \u003cem\u003ePTR\u003c/em\u003e and \u003cem\u003eOPT7\u003c/em\u003e in the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant could contribute to the marked increase in Fe concentration in the grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to Fe, the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant accumulated high Mn and Ni levels in the shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and Zn and Cu in the grains (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This can be explained by the fact that DMA and NA not only bind to Fe but also have an affinity for Mn\u003csup\u003e2+\u003c/sup\u003e, Ni\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, and Zn\u003csup\u003e2+\u003c/sup\u003e, thereby facilitating the translocation and transport of these essential transition metals in shoots (Murakami et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Mari et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Curie et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In contrast, the contents of non-essential Cd, Pb, and Co were not increased in the shoots of the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Although recent reports indicate that the overexpression of NAS in yeasts or Arabidopsis enhances Cd mobility (Hollmann et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), we did not obtain any data indicating that the \u003cem\u003ehrz1\u003c/em\u003e mutation enhances Cd uptake. This is likely because the primary Cd uptake pathway in rice is OsNRAMP5, which transports Cd\u0026sup2;⁺, the predominant chemical form in aerobic paddy fields (Ishikawa et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sasaki et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, we confirmed the absence of increased expression of any transporters associated with heavy metal transport in the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), including \u003cem\u003eOsZIP\u003c/em\u003es (Zn and Cd transporters), \u003cem\u003eOsNramp5\u003c/em\u003e (Cd, Pb, and Mn transporters), \u003cem\u003eOsHMA3\u003c/em\u003e (vacuolar Cd transporter), and \u003cem\u003eFPN1\u003c/em\u003e (Ni and Co transporters; Kaur et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kan et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results revealed that the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation could serve as a beneficial target gene site for improvement in Fe biofortification, simultaneously increasing the Zn and Cu content in grains while preventing the absorption of harmful elements.\u003c/p\u003e\u003cp\u003eWhen developing Fe-biofortified crops using \u003cem\u003ehrz\u003c/em\u003e/\u003cem\u003ebts\u003c/em\u003e mutations, considering the risk of Fe toxicity in plants is essential. Unlike rice, most other plant species are sensitive to excess Fe. Indeed, severe Fe toxicity with defective embryo development and leaf necrosis could occur because of increased amounts of Fe in the shoots of Arabidopsis \u003cem\u003ebts\u003c/em\u003e mutants and a pea \u003cem\u003eHRZs/BTS\u003c/em\u003e ortholog mutant \u003cem\u003edgl\u003c/em\u003e (\u003cem\u003edegenerate leaves\u003c/em\u003e) grown under normal soil conditions (Welch and Larue \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Selote et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Harrington et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, for crops that are not tolerant to Fe toxicity, it may be challenging to confer a trait that significantly increases Fe content in shoots, such as the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant. Previous reports have indicated that \u003cem\u003ehrz1\u003c/em\u003e knockdown rice is more prone to Fe toxicity when grown under Fe-excess conditions (Aung et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kobayashi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the present study, although no adverse effects on yield due to excess Fe were observed in RILs with the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation under the soil conditions used (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), bronzing, an indicator of excess Fe, occasionally appeared on the leaf tips during later growth stages. Therefore, enhancing excess Fe tolerance in rice plants harboring the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutation through breeding or agronomic approaches will contribute to further dissemination and practical applications of Fe-biofortified rice in the future. In this context, recently reported rice lines possessing excess Fe tolerance traits (Rosdianti et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) may be a promising option for breeding materials.\u003c/p\u003e\u003cp\u003eAlthough various attempts have been made to develop Fe-biofortified rice using the transgenic strategy, its widespread commercialization is limited by the difficulty in obtaining approval or acceptance of such genetically modified plants in various countries. In this context, mutation breeding with a DNA marker for the \u003cem\u003etetsu\u003c/em\u003e-type mutation is an effective strategy to facilitate the practical application of Fe biofortification in non-genetically modified rice. Here, we succeeded in conferring rice with increased Fe and other nutritional, polyphenol, and phenolic components (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The RILs generated in this study can be used in regular edible rice or processed rice products.\u003c/p\u003e\u003cp\u003eIn summary, we successfully established Fe-biofortified rice lines that exhibited normal growth, fertility, and various brown rice traits by breeding a \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant. These results confirmed that the identified novel \u003cem\u003eHRZ1\u003c/em\u003e mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars. Currently, we are conducting safety tests in animals to confirm their efficacy and anticipate that these RILs will become widely available.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e\u003cp\u003eT65 (accession no. T0504) seeds were provided by the National Institute of Genetics (Japan). The T65 MNU-induced rice mutants (TCM mutants) were generated by T.K. (Kyushu University) and provided to T.F. or A.S. through the National BioResource Project (MEXT, Japan). \u0026lsquo;Asamurasaki\u0026rsquo; seeds were purchased from a Japanese seed company (Noguchi Seeds Co., Saitama, Japan). For the primary screening of the TCM mutants, 128-cell plug trays (Takii Seed Co., Kyoto, Japan) were used for cultivation with commercial soil at pH 5.0, N:P:K\u0026thinsp;=\u0026thinsp;0.8:2.0:1.0 (Kumiai Chemical Industry Co., Tokyo, Japan). The mineral content of the soil was determined as described previously (Uraguchi et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The seeds were disinfected by soaking in 60\u0026deg;C water for 10 min, followed by watering for 3 days to promote germination. For each seedling tray, eight plants of the T65 and 90 lines of TCM mutants were sown, totaling 2887 TCM lines. The plants were cultivated for 21 days under natural light in a greenhouse. For the cultivation of \u003cem\u003etetsu\u003c/em\u003e mutant selected from the seed pool of TCM1587 was grown on soil in an air-conditioned greenhouse (26\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C). For yield surveys of the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant and RIL lines, plants were grown in soil in a controlled greenhouse under natural light and supplemental light with a light intensity of more than 400 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e during the day, with a 14 h light (28\u0026deg;C)/10 h dark (25\u0026deg;C) cycle. For the alkaline soil experiments, young seedlings were pre-cultured in soil (pH 5.0) for 38 days and then transferred to soil with pH 5.0 or 8.7.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eXylem sap collection for determining metal concentrations\u003c/h3\u003e\n\u003cp\u003eXylem sap collection was performed according to previous reports (Uraguchi et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Yamamura et al. \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) with minor modifications. Briefly, the aboveground portion was cut at a point 3 cm above the ground using a razor blade. A 1-mL chip filled with small pieces of quartz wool was placed over the cut surface, and the exuding xylem sap was absorbed into the quartz wool over a 3-hour period. The 1-mL chip containing quartz wool was inverted and placed into a 2-mL tube, and the xylem sap was collected by centrifugation at 3,500 rpm for 2 min. The collected xylem sap was weighed, stored at \u0026minus;\u0026thinsp;80\u0026deg;C until analysis.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAnalysis of metal concentration in rice plants\u003c/h2\u003e\u003cp\u003eIn the metal analysis of shoots during primary screening, up to 100 mg of homogenized dry rice leaves was added to a Teflon-coated centrifuge tube, and 3 mL of a mixture of concentrated nitric acid and hydrogen peroxide (3:1) was added. The sample was placed in a heat block and heated at 80\u0026deg;C for 1 h and 100\u0026deg;C for 20 min, then dissolved by a vortex mixer. The reaction was continued at 150\u0026deg;C for 4 h, with the acid mixture added as needed to complete the reaction. After drying, the sample was resuspended in 0.08 N nitric acid containing 2 ppb indium (In) as an internal standard. For secondary screening and subsequent analyses, 50 mg or less of dried rice leaf powder or 5\u0026ndash;10 grains of brown rice were dissolved in 5 mL of concentrated nitric acid. The collected xylem sap was diluted with a fixed volume of 1 N nitric acid. The supernatants of the inorganic element extraction solutions were analyzed using inductively coupled plasma mass spectrometry (ICP-MS, model SPQ 9700; SII Nano Technology, currently Hitachi High-Tech Science, Inc.) for the 1st screening and a furnace atomic absorption spectrophotometer (AA-6300 with GFA-EX7i, Shimadzu, Tokyo, Japan) for the 2nd screening as described (Saito et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eIron staining of rice seeds using Perls’ staining\u003c/h3\u003e\n\u003cp\u003ePerls\u0026rsquo; staining was performed by adding 1 mL of Perls\u0026rsquo; staining solution (4% v/v HCl and 4% w/v K-ferrocyanide) (Roschzttardtz et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) to 7\u0026ndash;8 cut brown rice grains, followed by staining for 90 min. After washing with ultrapure water, images were captured using a stereomicroscope. To prepare longitudinal brown rice sections, one side of the brown rice was adhered to the surface of the cap of a 2 mL screw tube using instant adhesive (Aron α, Quick-Setting Multi-Purpose Extra Type, Konishi Co., Ltd., Osaka, Japan). The 2-mL tube cap, with the rice-adhered side facing upward, was then fixed to the sample tray of a microtome (DTK-1000, DOSAKA EM Co., Ltd., Kyoto, Japan) using the same instant adhesive. A razor blade was aligned with the center of the rice germ, and the sample was cut in half using a microtome set to a vibration frequency of 10 Hz (maximum value) and a cutting speed of 2\u0026ndash;3 mm/s. The side without the adhesive was used for Perls\u0026rsquo; staining.\u003c/p\u003e\n\u003ch3\u003eMeasurement of SPAD values in leaves\u003c/h3\u003e\n\u003cp\u003eThe chlorophyll concentration indices of the leaves were measured using a mobile device (SPAD-50-Plus; KONICA MINOLTA JAPAN, Inc., Tokyo, Japan), and the average of three central areas of the latest expanded leaves was measured for each plant.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eSoil analysis\u003c/h2\u003e\u003cp\u003eSoil pH was measured by placing 10.0 g of air-dried soil in a 100-mL glass container, adding 100 mL of pure water, and shaking at 25\u0026deg;C and 135 rpm for 24 h until the pH of the alkaline soil solution reached equilibrium. The pH of the supernatant was then measured. Soil-soluble Fe content was determined according to a well-established method (Lindsay and Norvell \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Briefly, a 20-mL diethylenetriamine-N,N,N',N'',N''-pentaacetic acid (DTPA) extraction solution (5 mM DTPA, 0.1 M triethanolamine hydrochloride, 10 mM calcium chloride, adjusted to pH 7.3 with NaOH) was added to 10.0 g of air-dried soil. The mixture was shaken at 25\u0026deg;C and 135 rpm for 2 h. The supernatant was filtered through No. 2 filter paper (Advantech Co., Ltd., Tokyo, Japan), and nitric acid was added to achieve a final concentration of 1%. The DTPA-extracted Fe was then measured using furnace atomic absorption spectroscopy (AA-6300 with GFA-EX7i, Shimadzu, Tokyo, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eData collection procedures for GRAS-Di\u003c/h2\u003e\u003cp\u003eA total of 97 individuals from the F\u003csub\u003e2\u003c/sub\u003e population and their parents were used for GRAS-Di analysis (Toyota Motor Corporation, Tokyo, Japan). First, germinated seeds were grown in soil for 16\u0026ndash;18 days, and then the shoots were cut with a razor blade. The Fe content of the shoots was analyzed in advance. After approximately 40 days, new shoots regenerated from the remaining plant stems. Based on the Fe content values, 54 high-Fe F\u003csub\u003e2\u003c/sub\u003e individuals and 50 low-Fe F\u003csub\u003e2\u003c/sub\u003e individuals, or 3 individuals from each parent, were selected. Healthy and most recently expanded leaves were collected, washed, and immediately frozen in liquid nitrogen. The leaves were then ground to a fine powder using a mortar and pestle under liquid nitrogen, and genomic DNA was extracted using a DNeasy Plant Mini Kit (69104; QIAGEN). GRAS-Di libraries were constructed using a NovaSeq 6000 S4 reagent kit. Libraries were sequenced using an Illumina NovaSeq 6000 (Sequence Mode: 2\u0026times;150; Flow Cell Type: S4; Illumina, San Diego, CA, USA). Genotyping was conducted using 38,950 dominant single-dose markers generated using GRAS-Di software ver. 1.0.5. (TOYOTA, Aichi, Japan). Sequence reads derived from sequence adapters and those with low sequence quality were excluded from data analysis. Among the resulting 14,757 amplicons, those showing clear amplification differences between the parents were selected. As a result, 1,068 amplicons positive only in the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant were designated as C1, and 1,022 amplicons positive only in \u0026lsquo;Asamurasaki\u0026rsquo; were designated as C2, yielding a total of 2,090 dominant GRAS-Di markers. The markers obtained were used to determine candidate positions in the reference genome sequence.\u003c/p\u003e\u003cp\u003e\u003cb\u003eWhole genome sequencing analysis of T65 and\u003c/b\u003e \u003cb\u003etetsu(hrz1)\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFive WT (T65) and eight \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant plants exhibiting a high-Fe phenotype were cultivated for 1 month after sowing. Genomic DNA was extracted from young leaves using the DNeasy Plant Mini Kit (69104; QIAGEN). Genomic DNA from each individual was mixed in a single tube to ensure equal amounts, resulting in bulk DNA samples for T65 and the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutants. Bulk DNA samples were analyzed using a Genomic DNA ScreenTape Assay (Agilent Technologies, Santa Clara, CA, USA) to determine the DNA Integrity Number (DIN), which was approximately 7, confirming the high integrity of genomic DNA. Genome shotgun analysis was performed using HiSeq X, with a read length of 2 \u0026times; 150. Data analysis involved cleaning the reads using Trimmomatic software (ver. 0.39) and mapping them to the reference sequence using BWA (ver. 0.7.17). The reference sequence used was \u0026ldquo;\u003cem\u003eOryza sativa\u003c/em\u003e, IRGSP-1.0.\u0026rdquo; Both samples contained more than 200 Mb of reads, with 99.8% of the reads mapped. Additionally, over 90% of mapped reads covered the reference sequence at 50\u0026times; depth, yielding high-precision sequence information. Picard tools (ver. 1.111) was used to remove the duplicate reads. Finally, we listed the bases that did not match T65 and the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The identified \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutation sites were confirmed using Sanger sequencing.\u003c/p\u003e\u003cp\u003e\u003cb\u003edCAPS method to detect the mutation in\u003c/b\u003e \u003cb\u003eHRZ1\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo detect \u003cem\u003etetsu\u003c/em\u003e-type mutations in \u003cem\u003eHRZ1\u003c/em\u003e, we created primers based on dCAPS Finder 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://helix.wustl.edu/dcaps/dcaps.html\u003c/span\u003e\u003cspan address=\"http://helix.wustl.edu/dcaps/dcaps.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Neff et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), setting the value for mismatches to 1. Among the lists of candidate primers, we selected the following primer sets that can cleave the objective PCR products with HincII: 5'-CTA TTG ATG GTC AGG TTG AAA GGC ATC CCA TAG ATG AGA TTC TGT GTT G-3' (49 bp) for the forward primer and 5'-AAC CTG AAT ACA CTA AGA GAA AGG T-3' (25 bp) for the reverse primer. Genomic DNA for PCR was extracted based on conventional methods (Edwards et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Scissored young rice leaves less than 1 cm in length were crushed with two stainless steel beads in a 2-mL tube, and the supernatant was collected and purified using the isopropanol precipitation method. More detailed information regarding the dCAPS method is provided in Fig. S6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eRNA-Seq analysis\u003c/h2\u003e\u003cp\u003eFrom the F\u003csub\u003e3\u003c/sub\u003e population that was backcrossed T65 with the \u003cem\u003etetsu(hrz1)\u003c/em\u003e mutant, homozygous WT (\u003cem\u003eHRZ\u003c/em\u003e+/+) and \u003cem\u003etetsu\u003c/em\u003e (\u003cem\u003ehrz1-/-\u003c/em\u003e) plants were selected. Leaves from two individuals were combined into one sample, and six individuals (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) were analyzed. The plants were grown in a greenhouse at 26\u0026deg;C under natural light for 60 days after sowing in culture soil (Bonsol No. 1; Sumitomo Chemical, Tokyo, Japan), and the youngest maximum expanded leaves (sixth leaf) of the main culms were used for analysis. Approximately 100 mg of the frozen leaf powder that had been ground in a mortar and pestle was transferred to a 2-mL tube (TM-626; TOMY MEDICO. Ltd., Tokyo, Japan) containing the RNA extraction solution of an RNeasy Plant Mini Kit (74904; QIAGEN), and crushed using two stainless steel beads (SUB-50, 4.8 mm φ; TOMY MEDICO. Ltd.) in a bead-crushing device (Micro Smash TM MS-100; TOMY MEDICO. Ltd.) at 4,000 rpm for 100 s. Genomic DNA was removed through DNase treatment, according to the standard protocol for the RNeasy Plant Mini Kit. cDNA was prepared by targeting RNA with a poly A tail using the SMART-Seq v4 Ultra Low Input Kit (#634888; Clontech Laboratories, Inc., Mountain View, CA, USA). A Nexera XT DNA Library Prep kit (Illumina) was used to create a cDNA library, and at least 80\u0026nbsp;million reads were sequenced per sample using NovaSeq (Illumina). Data analysis was performed using the DRAGEN Bio-IT Platform (version 3.7.5; Illumina). The read sequences obtained from the sequencing analysis were mapped to the reference genome sequence (\u003cem\u003eOryza sativa\u003c/em\u003e IRGSP-1.0), and approximately 99% of all reads were identified. The expression levels of the genes and transcripts were calculated based on the positional information obtained from the mapping and gene definition files.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of total anthocyanin in rice grains\u003c/h2\u003e\u003cp\u003eAnthocyanin content was measured according to Watanabe et al. (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Approximately 100 mg of brown rice was mixed with 1 mL of anthocyanin extraction solvent (methanol: water: trifluoroacetic acid\u0026thinsp;=\u0026thinsp;40:60:0.5), and the mixture was incubated at 37\u0026deg;C overnight. The mixture was then ground four times using a bead-crushing device (Micro Smash TM MS-100, TOMY MEDICO. Ltd.) with three zirconia beads (φ3 mm) at 4,000 rpm for 120 s, with ice cooling applied each time. Centrifugation was performed at 15,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 min at 4\u0026deg;C, and the supernatant was collected. The supernatant was diluted 5-fold with the extraction solvent, centrifuged at 15,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 5 min at room temperature to remove insoluble residues, and the absorbance was measured at 525 nm. Total anthocyanin content was calculated based on the molar extinction coefficient of cyanidin-3-glucoside (Singh et al., 2022).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of phenolic acids in rice grains\u003c/h2\u003e\u003cp\u003eA total of 600 \u0026micro;L methanol containing 50 \u0026micro;M internal standards (L-methionine sulfone, 2-morpholinoethanesulfonic acid, and \u003csup\u003e13\u003c/sup\u003eC\u003csub\u003e6\u003c/sub\u003e-glucose) was added to 45\u0026ndash;50 mg of brown rice powder and mixed using a bead-crushing device under cooling conditions (1,500 rpm, 120 s \u0026times; 2 times). Thereafter, 600 \u0026micro;L of Milli-Q water was added, stirred, and centrifuged (2,300 \u0026times; \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C, 5 min). The supernatant was transferred to an ultrafiltration tube (UltraFree-MC-PLHCC, 5 kDa ultrafiltration membrane, Human Metabolome Technologies, Inc. Yamagata, Japan), and centrifuged (9,100 \u0026times; \u003cem\u003eg\u003c/em\u003e, 4\u0026deg;C, 120 min). Subsequently, the filtrate was dried, dissolved in an aliquot of Milli-Q water, and used for measurements. The sample was analyzed using an Agilent CE-TOFMS system (Agilent Technologies) with a fused silica capillary (i.d. 50 \u0026micro;m \u0026times; 80 cm) in anion mode. Peaks detected using CE-TOFMS were automatically extracted using the automatic integration software MasterHands ver.2.19.0.2. The relative area values were calculated by dividing the area of the target peak by the product of the area of the internal standard and sample amount. Based on the m/z and MT values, all substances registered in the metabolite library were compared to identify phenolic compounds. The acceptable error for the search was set to \u0026plusmn;\u0026thinsp;0.5 min for MT and \u0026plusmn;\u0026thinsp;10 ppm for m/z. For the obtained peaks, the relative area value ratios for each group were calculated and \u003cem\u003et\u003c/em\u003e-tests were performed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eComparisons between two groups were performed using Student\u0026rsquo;s t-test, and multiple group comparisons were analyzed by one-way ANOVA followed by Tukey\u0026rsquo;s test. All statistical analyses were conducted using Microsoft Excel and Statistics Kingdom (2017): ANOVA Calculator (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.statskingdom.com/180Anova1way.html\u003c/span\u003e\u003cspan address=\"https://www.statskingdom.com/180Anova1way.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMNU; N-methyl-N-nitrosourea, T65; Taichung-65, GRAS-Di; Genotyping by Random Amplicon Sequencing-Direct, HRZ1/2; Hemerythrin (or Hemerythrin) motif-containing Ring Zinc-finger protein 1/2, RIL; Recombinant Inbred Line, IRT1/2; Iron-Regulated Transporter 1/2, DMA; 2\u0026rsquo;-deoxymugineic acid, NAS; Nicotianamine Synthase, NA; nicotianamine, NAAT; Nicotianamine Aminotransferase, DMAS; Deoxymugineic Acid Synthase, TOM1; Transporter of Mugineic acid family phytosiderophores 1, YS1; Yellow Stripe 1 transporter, YSL; YS1-Like transporter, IDS2/3; Iron Deficiency Specific clone no. 2/3 (DMA dioxygenases), IDEF1/2; Iron-Deficiency-responsive Elements-binding transcription Factor 1/2, OsFRDL1; Ferric Reductase Defective Like 1, PEZ1/2; Phenolics Efflux Transporter 1/2, ENA; Efflux transporter of NA, VMT; Vacuolar Mugineic acid Transporter, VIT1/2; Vacuolar Iron Transporter 1/2, FER; Fe-storage protein ferritin, MIT; Mitochondrial Iron Transporter, OsIRO2/3; Iron-regulated transcription factor 2/3, bHLH; basic Helix-Loop-Helix, PYE; POPYE, BTS; BRUTUS, HHE; histidine, histidine, and glutamic acid-containing domain, \u0026nbsp;HORZ1; Hemerythrin motif-containing protein without RING- and Zn-finger 1, IMA1/2; IRON MAN/ FE uptake inducing Peptide, DTPA; Diethylenetriamine-N,N,N\u0026apos;,N\u0026apos;\u0026apos;,N\u0026apos;\u0026apos;-pentaacetic acid\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eEthics approval and consent to participate\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eConsent for publication\u003c/h3\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch3\u003eAvailability of data and material\u003c/h3\u003e\n\u003cp\u003eThe datasets supporting the conclusions of this study are included in this article and its additional files.\u003c/p\u003e\n\u003ch3\u003eCompeting interests\u003c/h3\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch3\u003eFunding\u003c/h3\u003e\n\u003cp\u003eThis work was supported by the following projects raised by the Research Institute of Tokyo University of Agriculture [Strategic Research Project for 2015-2016, Dean-led Research Project for 2018-2020, and Kome (rice) Project for 2021-2025] to A.S.\u003c/p\u003e\n\u003ch3\u003eAuthors\u0026apos; contributions\u003c/h3\u003e\n\u003cp\u003eA.S. performed the initial screening of TCM mutant lines, designed and conducted subsequent experiments, analyzed data, and was the major contributor to manuscript writing. T.Kumamaru. generated the TCM mutants. J.K. and M.S. conducted the secondary screening of candidate lines. K.N. and H.I. made initial crosses between the \u003cem\u003etetsu\u003c/em\u003e(\u003cem\u003ehrz1\u003c/em\u003e) mutant and other rice varieties. A.H., M.K., and K.S. developed and selected rice RILs. S.K., S.Nakayama, and N.K. performed yield surveys and elemental analyses. N.K. also cultivated plants and extracted DNA for GRAS-Di analysis. H.N. and A.K. performed the dCAPS analysis. R. Shimokawa and S.M. conducted alkaline soil tests. S.Nishino, T.Kobayashi, K.Y., R.Sugano, and H.H. carried out generation-advancement cultivation and Fe analysis of Fe-biofortified lines. T.O., Y.S., T.Kumamaru, S.U., T.F., and K.H. provided essential guidance and supported experiments, discussions, and manuscript revision.\u003c/p\u003e\n\u003ch3\u003eAcknowledgements\u003c/h3\u003e\n\u003cp\u003eThe rice seeds of \u003cem\u003eO. sativa\u003c/em\u003e cv. The T65 (T0504) cells were provided by the National Institute of Genetics (Japan) to A.S. We are grateful to Kayoko Aizawa for the excellent technical assistance during primary screening at the University of Tokyo. We also thank all the members of the research projects at TUA, especially Yumi Aizawa, Tsukasa Suzuki, Hiroyuki Oshima, Hirofumi Inoue, Daiki Oka, Akinobu Kajikawa, Haruko Noguchi, Yoshimasa Tsujii, Hiroko Suzuno, Toshimori Kadokura, Kazuhiro Homma, Yuji Yamamoto, Kazuko Yamaguchi-Shinozaki, and Takuji Sasaki. The authors acknowledge the use of a rice paddy field in the Kanto region of Japan for the cultivation trials, supported by Taku Kato of TUA and the field owners Mr. Mrs. Hideo and Sachiko Noji.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAoyama T, Kobayashi T, Takahashi M, Nagasaka S, Usuda K, Kakei Y, Nishizawa NK (2009) OsYSL18 is a rice iron (III)\u0026ndash;deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints. 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BMC Plant Biol 10(1):166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2229-10-166\u003c/span\u003e\u003cspan address=\"10.1186/1471-2229-10-166\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Fe-biofortified plant, Fe uptake and accumulation, Fe-deficiency anemia, HRZ1, Rice (Oryza sativa), GRAS-Di","lastPublishedDoi":"10.21203/rs.3.rs-7955540/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7955540/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIron (Fe) deficiency anemia is a significant public health problem worldwide. The development of Fe biofortification in staple food crops, such as rice, through non-transgenic methods is highly anticipated to enhance broad applicability. In this study, we isolated a high Fe-accumulating mutant (\u003cem\u003etetsu\u003c/em\u003e) from an \u003cem\u003eN\u003c/em\u003e-methyl-\u003cem\u003eN\u003c/em\u003e-nitrosourea (MNU) mutagenized \u0026lsquo;Taichung-65 (T65)\u0026rsquo; rice population. The \u003cem\u003etetsu\u003c/em\u003e mutant accumulated more than 3-fold higher levels of Fe and significantly higher levels of manganese (Mn) and nickel (Ni) in the shoot than the wild-type T65, whereas the levels of toxic heavy metals such as cadmium (Cd), lead (Pb), and cobalt (Co) were comparable to those of the wild-type. In both polished and brown rice of the \u003cem\u003etetsu\u003c/em\u003e mutant, Fe increased by approximately 2-fold, and Zn and Cu also significantly increased compared with those in T65. Perls\u0026rsquo; staining revealed that Fe localization in rice grains was not limited to the outer layers and scutellum, but also extended into the endosperm of the \u003cem\u003etetsu\u003c/em\u003e mutant. Concomitant with high Fe accumulation, the \u003cem\u003etetsu\u003c/em\u003e mutant showed remarkable tolerance to alkaline Fe-deficient soil conditions. Genotyping by Random Amplicon Sequencing-Direct (GRAS-Di) analysis revealed a novel nonsense mutation in the \u003cem\u003eHemerythrin motif-containing Ring Zinc-finger protein 1\u003c/em\u003e (\u003cem\u003eHRZ1\u003c/em\u003e) gene in the \u003cem\u003etetsu\u003c/em\u003e genome, which is known to govern the negative regulation of the Fe deficiency response and is crucial for normal development. The homozygous \u003cem\u003etetsu\u003c/em\u003e mutation leads to a substantial increase in shoot Fe content, alongside the upregulation of several genes related to Fe uptake and translocation, without causing serious adverse effects on growth. To utilize this novel mutation in Fe-biofortified rice breeding, we created recombinant inbred lines (RILs) derived from crosses between the \u003cem\u003etetsu\u003c/em\u003e mutant and \u0026lsquo;Asamurasaki,\u0026rsquo; a nutrient-rich black rice cultivar. During the breeding process, we successfully selected RILs that exhibited normal growth and fertility, resulting in the development of non-transgenic Fe-biofortified rice lines with various waxy/glutinous properties and polyphenol content in brown rice for versatile applications. These results indicate that the identified novel \u003cem\u003eHRZ1\u003c/em\u003e mutation is a valuable target for engineering non-transgenic Fe-biofortified rice cultivars with various beneficial traits.\u003c/p\u003e","manuscriptTitle":"Establishment of a non-transgenic iron-biofortified rice line using a novel HRZ1 mutation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 14:21:16","doi":"10.21203/rs.3.rs-7955540/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-28T01:33:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-22T17:43:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"328455523272830187032494876423446408891","date":"2025-12-04T16:40:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91733171675147970284578404138526465693","date":"2025-12-03T11:13:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62958421308514666972709887423403120571","date":"2025-12-02T00:32:54+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T09:34:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"196830281867302552066007162623932926185","date":"2025-11-13T01:51:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-03T00:32:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-27T11:02:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-27T11:01:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2025-10-27T07:42:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fa3f6e8a-85db-49ec-a0c5-4821c73c9679","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-16T16:02:58+00:00","versionOfRecord":{"articleIdentity":"rs-7955540","link":"https://doi.org/10.1186/s12284-026-00897-6","journal":{"identity":"rice","isVorOnly":false,"title":"Rice"},"publishedOn":"2026-03-13 15:58:15","publishedOnDateReadable":"March 13th, 2026"},"versionCreatedAt":"2025-11-12 14:21:16","video":"","vorDoi":"10.1186/s12284-026-00897-6","vorDoiUrl":"https://doi.org/10.1186/s12284-026-00897-6","workflowStages":[]},"version":"v1","identity":"rs-7955540","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7955540","identity":"rs-7955540","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}