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. 2019 Jul 22;181(1):276–288. doi: 10.1104/pp.19.00598

A Vacuolar Phytosiderophore Transporter Alters Iron and Zinc Accumulation in Polished Rice Grains1,[OPEN]

Jing Che 1, Kengo Yokosho 1, Naoki Yamaji 1, Jian Feng Ma 1,2,3
PMCID: PMC6716231  PMID: 31331995

Knockout of a vacuolar transporter for a phytosiderophore enhances iron and zinc accumulation in polished rice grains through increasing solubilization of these metals deposited in the node.

Abstract

Essential metals, such as iron (Fe) and zinc (Zn), in grains are important sources for seed germination and nutritional requirements, but the molecular mechanisms underlying their loading into grains are poorly understood. Recently, nodes in rice (Oryza sativa) were reported to play an important role in the preferential distribution of mineral elements to the grains. In this study, we functionally characterized a rice gene highly expressed in nodes, OsVMT (VACUOLAR MUGINEIC ACID TRANSPORTER), belonging to a major facilitator superfamily. OsVMT is highly expressed in the parenchyma cell bridges of node I, where Fe and Zn are highly deposited. The expression of OsVMT was induced by Fe deficiency in the roots but not in the shoot basal region and uppermost node. OsVMT localized to the tonoplast and showed efflux transport activity for 2′-deoxymugineic acid (DMA). At the vegetative stage, knockout of OsVMT resulted in decreased DMA but increased ferric Fe in the root cell sap. As a result, the concentration of DMA in the xylem sap increased but that of ferric Fe decreased in the xylem sap in the mutants. In the polished rice grain, the mutants accumulated 1.8- to 2.1-fold, 1.5- to 1.6-fold, and 1.4- to 1.5-fold higher Fe, Zn, and DMA, respectively, than the wild type. Taken together, our results indicate that OsVMT is involved in sequestering DMA into the vacuoles and that knockout of this gene enhances the accumulation of Fe and Zn in polished rice grains through DMA-increased solubilization of Fe and Zn deposited in the node.

Micronutrients such as iron (Fe) and zinc (Zn) in grains are not only required for the germination and early growth of plants (Lanquar et al., 2005) but also provide major nutritional sources for humans (Homo sapiens), especially for people who consume cereals as staple foods. However, more than two billion people are suffering from micronutrient deficiency, which is most prevalent in developing countries. Among micronutrients, deficiency of Fe and Zn is the most widespread nutritional deficiency in the world (Gernand et al., 2016). Rice (Oryza sativa) is a staple food for half of the world’s population; however, it contains low concentrations of Fe and Zn in the polished rice (Yang et al., 1998). Polished grains of popular rice cultivars have concentrations of approximately 2 μg g−1 Fe and 16 μg g−1 Zn (Trijatmiko et al., 2016), which is much lower than the biofortified rice targets: 13 μg g−1 Fe and 28 μg g−1 Zn to reach 30% of the human estimated average requirement (Bouis et al., 2011). Therefore, increasing Fe and Zn concentrations in rice grains through biofortification is expected to solve the chronic micronutrient malnutrition problems. However, the molecular mechanisms underlying the distribution of micronutrients to the grains are poorly understood.

There are two different sources for the transport of Fe and Zn to rice grain: retranslocation from other organs, such as old leaves, and uptake from the soil after flowering. Transporters involved in the redistribution of Fe and Zn from other organs to the grains have not been identified in rice, but several transporters involved in the uptake and distribution have been reported. Rice takes up both ferrous Fe (Fe2+) and ferric Fe (Fe3+) depending on growth conditions. In paddy soil, the major form of Fe is Fe2+, which is present at high concentration due to soil reduction (Ishimaru et al., 2006). Uptake of Fe2+ is proposed to be mediated by IRON-REGULATED TRANSPORTER1 (IRT1), a divalent metal ion transporter (Bughio et al., 2002). However, under upland conditions, Fe is present in oxidized insoluble Fe3+ compounds. To acquire this insoluble form, rice roots secrete 2′-deoxymugineic acid (DMA), a phytosiderophore, to chelate Fe3+. Secretion of DMA from the roots shows a distinct diurnal rhythm (Nishizawa and Mori, 1987; Negishi et al., 2002; Nozoye et al., 2014), which is mediated by TRANSPORTER OF MUGINEIC ACID1 (TOM1; Nozoye et al., 2011). The resulting Fe(III)-DMA complex is taken up by YELLOW STRIPE1-LIKE15 (OsYSL15; Inoue et al., 2009; Lee et al., 2009). After uptake, a portion of Fe is sequestered into the vacuoles by VACUOLAR IRON TRANSPORTER1 (OsVIT1) and OsVIT2 (Zhang et al., 2012), while the remaining portion is loaded into the xylem by unknown transporters, although FRD3-LIKE PROTEIN1 (OsFRDL1), a citrate transporter, is required for the efficient root-to-shoot translocation of Fe in rice (Yokosho et al., 2009). On the other hand, Zn uptake was previously suggested to be mediated by ZIP transporters, although the exact transporter has not been identified (Ramesh et al., 2003; Bashir et al., 2012). Sequestration of Zn to the root vacuoles is mediated by HEAVY METAL P-TYPE ATPASE3 (OsHMA3; Ueno et al., 2010; Cai et al., 2019), while its root-to-shoot translocation is mediated by OsHMA2 localized at the root pericycle cells (Yamaji et al., 2013b).

After heading, a portion of Fe and Zn taken up by the roots will be delivered to the grain. Distribution of mineral elements, including Fe and Zn, to the grains occurs in upper nodes, especially at node I, the uppermost node, which connects flag leaf and panicles. Rice nodes, including node I, have highly developed vascular systems, mainly consisting of enlarged vascular bundles (EVBs) and diffuse vascular bundles (DVBs; Yamaji and Ma, 2014, 2017). EVBs come from the two lower nodes and are connected to the leaf attached to the node, while DVBs start at the node and are connected to the upper two nodes or panicle. Therefore, an intervascular transfer of mineral elements from EVBs to DVBs is required for delivering mineral elements to the grains. Three steps for the intervascular transfer are required: unloading of a mineral nutrient from xylem flow in EVBs, transfer through the symplast or apoplast of a parenchyma cell bridge (PCB), and reloading to the xylem or phloem of a DVB (Yamaji and Ma, 2017). However, only a few transporters involved in the intervascular transfer of Fe and Zn have been identified. For Zn, two transporters (OsZIP3 [ZRT, IRT-LIKE PROTEIN3] and OsHMA2) are required for Zn distribution to the grain. OsZIP3 is localized at the xylem transfer cells in EVBs and is involved in unloading of Zn from the xylem of EVBs (Sasaki et al., 2015), while OsHMA2 is localized at the phloem region of both EVBs and DVBs and is responsible for loading Zn to the phloem of DVBs and EVBs (Yamaji et al., 2013b). On the other hand, transporters for intervascular transfer of Fe have not been identified, but OsFRDL1 is required for the distribution of Fe to panicles by releasing citrate to solubilize apoplastic Fe deposited in the PCB (Yokosho et al., 2016a). In addition, two members belonging to the rice YSL family, OsYSL2 and OsYSL9, are involved in Fe loading and distribution in developing rice grains, respectively (Ishimaru et al., 2010; Senoura et al., 2017).

To further understand the mechanisms underlying loading of Fe and Zn to rice grains, we have focused on transporter genes highly expressed in node I (Yamaji et al., 2013a). In this study, we functionally characterized one of them, OsVMT (VACUOLAR MUGINEIC ACID TRANSPORTER)/OsZIFL12 (ZINC-INDUCED FACILITATOR-LIKE12), belonging to a major facilitator superfamily (MFS). OsVMT showed the highest expression in node I, and the protein was localized to the tonoplast and transported DMA. Furthermore, knockout of this gene resulted in increased accumulation of Fe and Zn in the polished rice grain.

RESULTS

Cloning of OsVMT and Phylogenetic Analysis

The full length of the OsVMT/OsZIFL12 cDNA (Os12g0133100) was amplified by PCR using primers designed based on the rice sequence database (http://rapdb.dna.affrc.go.jp/). The open reading frame (ORF) showed an identical sequence with the database, which is composed of 1,509 bp, encoding a protein with 502 amino acids (Supplemental Fig. S1). OsVMT belongs to the ZIFL group of the MFS. A BLAST search on the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) showed 13 homologous genes in the rice genome (Supplemental Fig. S1). OsVMT shares 62%, 66%, and 86% identity, respectively, with OsTOM1 (OsZIFL4), OsTOM2 (OsZIFL5), and OsTOM3 (OsZIFL7), which were previously characterized (Supplemental Fig. S1; Nozoye et al., 2011, 2015; Ricachenevsky et al., 2011). They all contained the MFS signatures and antiporter signatures for the MFS proteins (Supplemental Fig. S1; Ricachenevsky et al., 2011). Furthermore, OsVMT also contained the conserved signatures (Cys motif and His motif) that are specific for the ZIFL proteins (Supplemental Fig. S1; Ricachenevsky et al., 2011).

Expression Patterns of OsVMT

We investigated the expression pattern of OsVMT in various organs at different growth stages of rice cultivated in paddy fields. OsVMT was highly expressed in the roots, although lower expression was also found in the shoots and shoot basal region at the vegetative growth stage (Fig. 1A). At the flowering stage, much higher expression of OsVMT was detected in the upper nodes, including nodes I, II, and III, peduncle, and rachis (Fig. 1A). At the grain-filling stage, OsVMT showed the highest expression in node I (Fig. 1A).

Figure 1.

Figure 1.

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Expression pattern of OsVMT. A, Relative expression of OsVMT in various organs at different growth stages. Rice was grown in a paddy field, and various organs were sampled at different growth stages. B, Response of OsVMT expression to metal deficiency in the roots. Rice was grown in a nutrient solution with or without Zn, Fe, manganese (Mn), or copper (Cu) for 1 week. C, Expression of OsVMT at the flowering stage. Rice was cultivated in a nutrient solution until flowering and then transferred to a solution with or without Fe for 1 week. The roots, shoot basal regions (1 cm from the root-to-shoot junction), and node I were sampled. D, Expression of OsVMT in different vascular tissues of node I separated by LMD at the milky stage. The expression level was determined by reverse transcription quantitative PCR (RT-qPCR). Expression relative to node I at the flowering stage (A), to the control roots (B), to root with Fe (C), and to PCB (D) is shown. HISTONE H3 and ACTIN were used as internal standards. Data are means ± sd of three biological replicates. Asterisks show significant differences when compared with the control in B, between −Fe and +Fe at each tissue in C, and compared with xylem (EVB) in D (P < 0.05 by Tukey’s test).

We investigated the response of OsVMT expression to a deficiency of essential metals in the roots. The expression of OsVMT was increased by Fe deficiency but slightly reduced by Mn, Zn, and Cu deficiency (Fig. 1B). Furthermore, Fe-induced expression was found in the roots but not in the basal nodes or node I at the reproductive stage (Fig. 1C).

We then investigated the tissue specificity of OsVMT expression within node I at the flowering stage using laser microdissection (LMD). OsVMT was highly expressed in the PCB (Fig. 1D). In the EVBs, expression of OsVMT was higher in the xylem region than in the phloem region (Fig. 1D).

Tissue Specificity of Localization of OsVMT

To investigate the tissue-specific expression of OsVMT, we generated transgenic rice lines carrying GFP under the control of the OsVMT promoter. Immunostaining with a GFP antibody showed higher GFP expression in the exodermis and endodermis of both root tip and basal root at the vegetative stage (Fig. 2, B, D, and E). At the reproductive stage, the signal was observed in the PCB, xylem region of EVB, and DVB of node I. The signal was stronger in the PCB than in the xylem region of EVB and DVB (Fig. 2, G and H). No signal was observed in the roots and node I of nontransgenic wild-type plants, indicating the specificity of the antibody (Fig. 2, A, C, and F). This result is consistent with that of LMD (Fig. 1D).

Figure 2.

Figure 2.

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Tissue specificity of OsVMT expression. Immunostaining with a GFP antibody was performed in transgenic rice carrying the OsVMT promoter fused with GFP (B, D, E, G, and H) or the wild type (A, C, and F) as a negative control in the roots (A–E) and node I (F–H). Red color indicates the GFP antibody-specific signal. Blue color indicates cell wall autofluorescence. Asterisks indicate cross talk of background autofluorescence from a particular cell wall. Yellow dotted boxes in D and G represent regions magnified in E and H, respectively. EVBs, xylem area of enlarged vascular bundle (XE), PCB, DVBs, and phloem area of enlarged vascular bundle (PE) in the node are shown. Bars = 50 μm.

Subcellular Localization of OsVMT

To determine the subcellular localization of OsVMT, the ORF of OsVMT was fused with GFP at both the C terminus and the N terminus under the control of the 35S promoter and then introduced into both the rice leaf protoplast and onion (Allium cepa) epidermal cells. GFP alone showed signal in the cytosol and nucleus in the rice protoplast (Fig. 3, A–D). However, both OsVMT-GFP and GFP-OsVMT were localized to the tonoplast in the rice protoplast (Fig. 3, E–L). When expressed in onion epidermal cells, OsVMT-GFP and GFP-OsVMT were also localized to the tonoplast (Supplemental Fig. S2).

Figure 3.

Figure 3.

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Subcellular localization of OsVMT in rice protoplasts. GFP alone (A–D) or OsVMT fused with GFP at the C terminus (E–H) and the N terminus (I–L) was transiently transformed into rice protoplasts by the polyethylene glycol method. GFP signal (A, E, and I), chlorophyll image (B, F, and J), bright field (C, G, and K), and merged image (D, H, and L) are shown. Bars = 10 µm.

Transport Activity of OsVMT

Since OsVMT is a homolog of OsTOM1, which is an efflux transporter of DMA in rice (Nozoye et al., 2011), we investigated its transport activity for DMA by the Xenopus laevis oocyte two-electrode voltage clamp system. There was no influx activity for DMA (Supplemental Fig. S3). By contrast, an efflux transport activity for DMA was detected; the outward current for DMA was more than two times higher in oocytes injected with OsVMT complementary RNA (cRNA) than the control (water injection; Fig. 4A). No efflux transport activity for nicotianamine (NA) or the Fe(III)-DMA complex was detected (Fig. 4A). Furthermore, we directly determined the efflux of DMA from oocytes using HPLC. The efflux activity for DMA was significantly higher in the oocytes injected with OsVMT cRNA than in oocytes injected with water at 1 and 3 h after DMA injection (Fig. 4B). These results consistently show that OsVMT is an efflux transporter for DMA rather than for NA and the Fe(III)-DMA complex.

Figure 4.

Figure 4.

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Efflux transport activity for DMA in X. laevis oocytes. A, The transport activities for DMA, NA, and Fe(III)-DMA were measured using the two-electrode voltage-clamp method. Water injection was used as a control. The oocytes expressing OsVMT cRNA were clamped at −100 mV. B, Efflux activity of DMA. Oocytes expressing OsVMT cRNA or not were injected with 50 nL of 50 mm DMA. The release of DMA from the oocytes was determined at 1 or 3 h by HPLC. The data are means ± sd of 10 independent oocytes injected with OsVMT cRNA or water (as a control). The different letters and asterisks show significant differences (P < 0.05 by Tukey’s test).

Phenotypic Analysis of OsVMT Knockout Lines at the Vegetative and Reproductive Growth Stages

To investigate the physiological role of OsVMT in plants, we used clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis to generate knockout lines of OsVMT. Two independent lines, vmt-a and vmt-t, which have a 1-bp insertion (adenine [A] or Tyr [T] insertion) at the targeted site, were obtained (Supplemental Fig. S4, A and B). RT-qPCR analysis showed almost no transcripts of OsVMT detected in the mutants (Supplemental Fig. S4C). Furthermore, the expression of OsTOM1 and OsTOM2 was not altered in the mutants (Supplemental Fig. S5, A and B).

There was no significant difference in growth at the vegetative growth stage between the wild type and two mutants when grown in a nutrient solution containing different Fe2+ concentrations ranging from 0 to 10 μm (Supplemental Fig. S6, A and B). There was also no difference in the growth between the wild type and the two mutants when grown at different Zn concentrations from 0.04 to 4 μm (Supplemental Fig. S6C).

When the mutants and the wild type were grown in the soil until ripening, no difference in the growth and grain yield was found between the lines (Fig. 5, A and B). The plant height, panicle number, grain number per panicle, fertility, and 1,000-grain weight were also similar between the wild type and the mutants (Fig. 5, C–G).

Figure 5.

Figure 5.

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Phenotypic analysis of vmt mutants at the reproductive stage. A and B, Growth performance (A) and panicle (B) of wild-type (WT) rice and vmt mutants at harvest. Bars = 10 cm. C to G, Plant height (C), panicle number (D), grain number per panicle (E), fertility (F) and 1,000-grain weight (G). Wild-type rice and two independent mutant lines were grown in pot soil until ripening. Data represent means ± sd (n = 3–4).

Ionomic Comparison between vmt Mutants and the Wild Type at Different Growth Stages

The Fe concentration of the roots, shoot basal region including basal node, and shoots was similar between the wild type and knockout mutants grown in a nutrient solution containing 0 and 0.2 μm Fe2+ (Fig. 6, A and B). However, the mutants accumulated lower Fe in the shoots at 2 and 10 μm Fe2+ (Fig. 6, C and D), although the concentration of Fe in the roots showed no difference between the wild type and mutants at either Fe condition (Fig. 6). Analysis of other metals showed that the concentrations of Cu, Zn, Mn, magnesium (Mg), phosphorus (P), and potassium (K) in the roots, shoot basal region, and shoots did not differ between the wild type and mutants (Supplemental Fig. S7).

Figure 6.

Figure 6.

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Fe concentration in the roots, shoot basal region, and shoot of vmt mutants and the wild type (WT) with different Fe treatments. Two-week-old seedlings of vmt mutants and the wild type were cultured in a nutrient solution containing 0 (A), 0.2 (B), 2 (C), or 10 (D) μm FeSO4 for 2 weeks. Data represent means ± sd (n = 3). Asterisks indicate significant differences between the wild type and vmt mutants in each tissue at either Fe condition (P < 0.05) using Tukey’s test.

By contrast, when the plants were exposed to different Zn concentrations, no difference in the Zn concentration of the roots, shoot basal region, and shoots was found between the mutants and the wild type at any Zn condition at the vegetative growth stage (Supplemental Fig. S8).

We also compared the concentrations of mineral elements in different organs between the wild type and vmt mutants grown in soil at harvest. A higher Fe concentration in node II, node I, leaf sheath, peduncle, rachis, and brown rice was found in vmt mutants than in the wild type (Fig. 7A). The mutants accumulated higher Zn in node I and brown rice (Fig. 7B). However, there was no difference in the concentrations of Mn and Cu in different organs (Supplemental Fig. S9).

Figure 7.

Figure 7.

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Comparison of Fe and Zn concentrations in different organs between the wild-type (WT) rice and two vmt mutants at the reproductive stage. A and B, Concentrations of Fe (A) and Zn (B) in different organs in the wild-type rice and two vmt mutants. C to E, Concentrations of Fe (C), Zn (D), and DMA (E) in polished rice grain. Wild-type rice and two independent mutant lines were grown in pots containing soil for 4 months. Data represent means ± sd (n = 3). Asterisks indicate significant differences (P < 0.05) between the wild type and vmt mutants in each tissue using Tukey’s test.

Further analysis of Fe and Zn concentrations in the polished grain showed that the vmt mutants accumulated 1.8 to 2.1 times higher Fe and 1.5 to 1.6 times higher Zn than the wild type (Fig. 7, C and D). The concentrations in the polished grain of vmt-t reached 8.8 mg kg−1 Fe and 34.5 mg kg−1 Zn (Fig. 7, C and D). Furthermore, vmt mutants contained a higher concentration of DMA in the polished grain than the wild type (Fig. 7E).

Fe Concentration in the Apoplastic and Symplastic Solutions of Rice Roots

To compare Fe partitioning in the roots of the wild type and vmt mutants, we determined Fe concentrations in the apoplastic and symplastic solutions (mostly from vacuoles) of different root segments. There was no difference in apoplastic Fe concentrations between the wild type and vmt mutants (Supplemental Fig. S10); however, the concentrations of Fe in symplastic solution was significantly higher in the mutants than in the wild type in both the root tips (0–1 cm from tip) and mature root regions (1–2 cm from tip). Furthermore, the Fe concentrations in the symplastic solution of both root tips and basal roots was seven to 10 times higher than that in the apoplastic solution (Supplemental Fig. S10).

Speciation of Fe and Concentration of Fe, DMA, and Citric Acid in Root Cell Sap and Xylem Sap

Since shoot Fe concentration was altered in vmt mutants at higher Fe conditions (Fig. 6, C and D), we compared the concentrations of different Fe forms (Fe2+ and Fe3+) in the xylem sap and root cell sap (mostly from vacuoles) between the wild type and knockout mutants. The total Fe concentration in the xylem sap of mutants was lower than that of the wild type (Fig. 8A), whereas that of Fe in the root cell sap of mutants was significantly higher than that of the wild type (Fig. 8B). Furthermore, speciation analysis showed that Fe2+ concentrations in the xylem sap and root cell sap were similar between the wild type and mutants, but the Fe3+ concentration in the xylem sap was lower, whereas that in the root cell sap was higher in the mutants compared with the wild type (Fig. 8, A and B). The concentration of DMA was significantly higher in the xylem sap but lower in the root cell sap in the mutants than in the wild type (Fig. 8, C and D). However, there was no difference in citrate concentrations in the xylem sap between the wild type and mutants (Fig. 8E).

Figure 8.

Figure 8.

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Fe speciation and concentrations of Fe, DMA, and citrate in the xylem sap and root cell sap. A and B, Concentrations of Fe2+ and Fe3+ in the xylem sap (A) and root cell sap (B). C to E, Concentrations of DMA in the xylem sap (C) and root cell sap (D) and of citrate in the xylem sap (E). Seedlings (18 d old) of both wild-type (WT) rice and vmt mutants were exposed to a nutrient solution containing 2 μm FeSO4. After 1 week, xylem sap and root cell sap were collected and subjected to determination of Fe2+ and Fe3+, DMA, and citrate. Data represent means ± sd (n = 3). Asterisks indicate significant differences (P < 0.05) between the wild type and vmt mutants using Tukey’s test.

Expression Profile of Genes Involved in Fe and Zn Transport

To investigate the effect of knockout of OsVMT in response to Fe deficiency, we compared the expression profiles of genes involved in Fe uptake (OsTOM1, OsTOM2, OsIRT1, OsIRT2, and OsYSL15) and Fe translocation (OsFRDL1). Consistent with previous studies (Ishimaru et al., 2006; Inoue et al., 2009; Lee et al., 2009; Yokosho et al., 2009; Nozoye et al., 2011, 2015), Fe deficiency did not affect OsFRDL1 expression but significantly increased the expression of OsTOM1, OsTOM2, OsIRT1, OsIRT2, and OsYSL15 (Supplemental Fig. S5). However, there was no difference in the expression of these genes between the wild type and vmt mutants in the absence and presence of Fe (Supplemental Fig. S5). The expression of OsHMA2 and OsZIP3 involved in Zn transport also did not differ between the wild type and vmt mutants (Supplemental Fig. S5).

DISCUSSION

OsVMT Is a Tonoplast-Localized Transporter for DMA

Our functional analysis of OsVMT showed that it is localized to the tonoplast (Fig. 3) and transports DMA rather than NA or the Fe(III)-DMA complex (Fig. 4). Among rice homologs of OsVMT, three members (OsTOM1, OsTOM2, and OsTOM3) have been functionally characterized. OsTOM1 and OsTOM2 also transport DMA (Nozoye et al., 2011, 2015); however, in contrast to OsVMT, both OsTOM1 and OsTOM2 are localized to the plasma membrane, although the subcellular localization of OsTOM3 has not been investigated (Nozoye et al., 2011, 2015). A homolog of OsVMT in Arabidopsis (Arabidopsis thaliana), AtZIF1, is also localized to the tonoplast (Haydon and Cobbett, 2007; Haydon et al., 2012); however, it does not transport DMA but does transport NA (Haydon et al., 2012). These results indicate that members in the ZIFL group have diverse subcellular localization and transport substrates depending on their role in transporting Zn and Fe, as described below.

OsVMT Is Responsible for Sequestering DMA to Vacuoles for Export of Fe(III)-DMA at the Vegetative Stage

TOM1 mediates the secretion of DMA from the roots to acquire insoluble Fe in soil (Nozoye et al., 2011), while TOM2 is involved in the mobilization of metals through the secretion of DMA into the vascular bundles for phloem and/or xylem metal loading (Nozoye et al., 2015). On the other hand, AtZIF1 is associated with tolerance to Zn toxicity (Haydon and Cobbett, 2007; Haydon et al., 2012). Our analysis showed that OsVMT has a distinct role from these homologs. At the vegetative growth stage, OsVMT was also highly expressed in both exodermis and endodermis of the roots (Fig. 2, B and D). Knockout of OsVMT resulted in increased Fe3+ and decreased DMA concentrations in the root cell sap (mostly from the vacuoles) at higher Fe concentrations (Fig. 8, B and D). In the xylem sap, the concentration of Fe3+ but not Fe2+ was decreased in the mutants, while that of DMA was increased (Fig. 8, A and C). Given that OsVMT is localized to the tonoplast (Fig. 3), these results suggest that OsVMT mediates the sequestration of DMA to the vacuoles of the root cells, forming the Fe(III)-DMA complex in the vacuoles, followed by exporting this complex from the vacuoles for subsequent translocation of this complex to the xylem. This is supported by the finding that a portion of Fe in the xylem sap is presented in the form of the Fe(III)-DMA complex (Kakei et al., 2009; Ariga et al., 2014). In the mutants, since DMA is not sequestered into the vacuoles, more DMA is loaded to the xylem (Fig. 8C). As a result, more Fe3+ accumulated in the root cell sap but less Fe3+ was found in the xylem sap (Fig. 8, A and B; Supplemental Fig. S9). However, since the dominant form of Fe in the xylem sap in rice is in the Fe-citrate complex (Kakei et al., 2009; Yokosho et al., 2009), knockout of OsVMT only slightly affected the total Fe concentration in the shoot (Fig. 6).

DMA is synthesized in some specific vesicles in both the roots and shoots (Nishizawa and Mori, 1987; Negishi et al., 2002; Nozoye et al., 2014), and its synthesis is enhanced by Fe deficiency (Higuchi et al., 2001). Under Fe deficiency, the expression of OsVMT was also up-regulated in the roots (Fig. 1C). This contributes to increased sequestration of DMA to the vacuoles for Fe homeostasis, although the exact role of OsVMT requires further investigation. On the other hand, under Fe-sufficient conditions, sequestration of DMA into vacuoles is responsible for Fe utilization. Since Fe is highly toxic to plant cells, its concentration in the cytosol must be kept low (Connolly and Guerinot, 2002). Therefore, once Fe (both Fe2+ and Fe3+) is taken up into the cells, it will be sequestered into the vacuoles. DMA will form a complex with Fe3+ in the vacuoles for subsequent export by an unidentified transporter for the Fe(III)-DMA complex.

In contrast to Fe, no difference in Zn concentration of root cell sap and xylem sap was found between the wild type and vmt mutants (Supplemental Fig. S11). This suggests that OsVMT is not involved in Zn homeostasis in the roots. In Arabidopsis, AtZIF1 mediates NA sequestration to the vacuoles and knockout of AtZIF1 results in increased sensitivity to high Zn (Haydon and Cobbett, 2007). In the rice genome, there are several other uncharacterized homologs of AtZIF1, and some of them may be involved in Zn homeostasis in the roots of rice.

Knockout of OsVMT Enhanced the Accumulation of Fe and Zn in Polished Rice Grains

Rice is the major calorie supplement for half of the world’s population but is a poor source of Fe and Zn due to their low concentrations in the grain and low bioavailability. Furthermore, 90% of Fe and 40% of Zn are lost during polishing. Therefore, enriching Zn and Fe concentrations in the polished rice grain and increasing their bioavailability are very important for people who suffer from micronutrient deficiency. Many attempts have been made to increase Fe and Zn concentrations in the polished rice grain by overexpressing genes involved in the synthesis of NA, Fe transport, and storage. For example, overexpression of OsNAS2 alone significantly increased both Fe (19 μg g−1) and Zn (76 μg g−1) concentrations in the polished rice grain (Johnson et al., 2011). Introduction of OsYSL2 (Fe-NA and Mn-NA transporter), HvNAS1 (barley [Hordeum vulgare] NA SYNTHASE1 gene), and SferH2 (soybean [Glycine max] FERRITIN gene) resulted in 4.4-fold Fe and 1.6-fold Zn increases in the polished grain of transgenic lines grown in the field (Masuda et al., 2012). An indica rice carrying OsNAS2 (for NA synthesis) and Sfer-H1 (for Fe storage) contained 15 μg g−1 Fe and 45.7 μg g−1 Zn in the polished grain (Trijatmiko et al., 2016). On the other hand, knockdown or knockout of ubiquitin ligase HRZ genes also increased Fe concentration in the shoots and grains by enhancing the expression of the genes involved in Fe acquisition (Kobayashi et al., 2013). In this study, we found that knockout of OsVMT resulted in significant increases in Fe and Zn concentrations in the polished rice grain without yield penalty (Figs. 5 and 7). These increases seem to be attributed to altered DMA partitioning, resulting in higher Fe and Zn accumulation in rice grains, especially in polished grains (Fig. 8, C and D). Since genes related to Fe and Zn transport did not differ between the wild type and vmt mutants (Supplemental Fig. S5), it is unlikely that the enhanced accumulation of Fe and Zn is indirectly caused by different Fe deficiency responses.

OsVMT is characterized by high expression in upper nodes, especially in PCBs (Fig. 2, G and H). In these tissues, Fe and Zn are also highly accumulated (Yamaguchi et al., 2012; Moore et al., 2014; Zhao et al., 2014; Yamaji and Ma, 2019). Since DMA chelates both Fe and Zn (Suzuki et al., 2008; Nishiyama et al., 2012), increased DMA in the cytosol and/or apoplast due to lack of vacuolar sequestration results in more Fe and Zn deposited in the PCBs to be solubilized in the mutant before loading to the grains through the phloem. This is supported by the result that more DMA is also found in the grain (Fig. 7E). Furthermore, there is a possibility that Fe(III)-DMA and Zn-DMA forms are easily transported to the endosperm compared with other forms. Therefore, although OsVMT is involved in vacuolar sequestration in both the roots and nodes, knockout of this gene yields different phenotypes due to different processes in the roots and nodes, as discussed above.

In conclusion, our results indicate that OsVMT is a tonoplast-localized transporter responsible for sequestration of a phytosiderophore (DMA) into the vacuoles. It is highly expressed in the PCBs of node I, and knockout of OsVMT increases Zn and Fe accumulation in the polished rice grains through increasing DMA-mediated solubilization of Fe and Zn deposited in the node. Combining OsVMT knockout with overexpression of other genes, such as OsNAS2, Sfer, and OsYSL2, may have the potential to further increase Fe and Zn accumulation in the polished rice grain. Furthermore, increased DMA in the polished grain may enhance the bioavailability of Fe and Zn.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Seeds of wild-type rice (Oryza sativa ‘Nipponbare’) and two CRISPR/Cas9 mutant lines (vmt-a and vmt-t) prepared as described below in "Generation of osvmt Knockout Lines by CRISPR/Cas9" were soaked in water at 30°C in the dark for 2 d and then transferred to a net floating on a 0.5 mm CaCl2 solution. On day 7, seedlings were transferred to a 3.5-L plastic pot containing one-half-strength Kimura B solution (Ma et al., 2002). The pH of this solution was adjusted to 5.6, and the nutrient solution was renewed every 2 d. Plants were grown in a closed greenhouse at 25°C to 30°C under natural light. At least three replicates were performed for each experiment.

Cloning the Full Length of OsVMT

To clone the full-length ORF of OsVMT, total RNA was extracted from rice roots (cv Nipponbare) using an RNeasy Plant Mini Kit (Qiagen) and then converted to cDNA using the protocol supplied by the manufacturer of ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo). The full-length ORF was amplified by PCR using primers listed in Supplemental Table S1, which were designed based on a putative cDNA clone (Os12g0133100) in the Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/) with putative translational start and stop sites. The amplified cDNA was cloned into pGEM-T vector (Promega), and the sequence was confirmed by a sequence analyzer (ABI Prism 3130; Applied Biosystems).

Phylogenetic Analysis

The alignment was performed with ClustalW using default settings (http://clustalw.ddbj.nig.ac.jp/), and the phylogenetic tree was constructed using the neighbor-joining algorithm with MEGA version 7.0 (Kumar et al., 2016). Bootstrap support was calculated (1,000 replications).

RT-qPCR

To investigate the expression pattern of OsVMT in different organs at different growth stages in rice grown in a paddy field, cDNA of rice (cv Nipponbare, the wild type) prepared in a previous study was used (Song et al., 2014). To examine the expression response to mineral deficiency, seedlings (4 weeks old) were grown in a nutrient solution with or without Zn, Fe, Mn, or Cu for 7 d. To compare the expression pattern between the wild type and vmt mutants in the roots, 2-week-old seedlings were exposed to 0 or 2 μm FeSO4 for 7 d. The roots were sampled for RNA extraction. Furthermore, at the flowering stage, the wild type cultivated hydroponically was transferred to a solution free of Fe. After 7 d, the roots, shoot basal region (1 cm from the root-to-shoot junction), and node I were sampled for RNA extraction. Tissue-specific expression in node I was investigated using samples prepared by LMD (Applied Biosystems Arcturus Laser Capture Microdissection System; Life Technologies) as described previously (Song et al., 2014).

Total RNA extraction and cDNA conversion were performed as described above. The expression level was determined by RT-qPCR using THUNDERBIRD qPCR mix (Toyobo) or SsoFast EvaGreen Supermix (Bio-Rad) on a real-time PCR machine (CFX384; Bio-Rad). ACTIN was used as an internal control for OsVMT expression in different vascular tissues of node I separated by LMD at the milky stage, while HISTONE H3 was used as an internal control for expression in other organs. Relative expression levels were calculated by the comparative Ct method. Three independent biological replicates were used for each treatment. The primer sequences used for investigating gene expression are shown in Supplemental Table S1.

Cellular Specificity of OsVMT Expression

To examine the tissue-specific expression of OsVMT, transgenic rice lines carrying OsVMT promoter fused with GFP were prepared. The promoter region of OsVMT (2.1 kb) was amplified from rice genomic DNA using primers listed in Supplemental Table S1. Then the promoter fragment was fused to the GFP fragment isolated from a pBluescript vector (Agilent). The fused DNA was inserted into the pPZP2H-lac vector (Fuse et al., 2001). The derived construct was transformed into calluses (cv Nipponbare) by Agrobacterium tumefaciens-mediated transformation (Hiei et al., 1994). Immunostaining with an anti-GFP antibody was performed in the roots and node I of the transgenic rice harboring pOsVMT-GFP and nontransgenic rice as a negative control as described before (Yamaji and Ma, 2007).

Subcellular Localization of OsVMT

Subcellular localization of OsVMT was investigated by introducing OsVMT-GFP and GFP-OsVMT fusions into rice protoplasts and onion (Allium cepa) epidermal cells. The ORF of OsVMT without or with the stop codon was amplified by PCR from rice (cv Nipponbare) root cDNA for OsVMT-GFP and GFP-OsVMT fusion, respectively. The ORF was inserted between the cauliflower mosaic virus 35S promoter and the GFP-NOS terminator in the pBluescript vector. The primers with the BspEI site are listed in Supplemental Table S1. Rice protoplasts were prepared from leaves of 2-week-old seedlings grown hydroponically and were used for transformation by the polyethylene glycol method as described previously (Chen et al., 2006). Introduction of the same fused genes into onion epidermal cells was performed as described previously (Yokosho et al., 2016b). GFP fluorescence was observed by laser confocal microscopy (LSM700; Carl Zeiss).

Transport Activity Assay in Xenopus laevis Oocytes

To investigate the transport activity of OsVMT in X. laevis oocytes, the ORF of OsVMT was amplified by RT-PCR using primers listed in Supplemental Table S1 and cloned into the BglII site of an X. laevis oocyte expression vector, pXbG-ev1 (Preston et al., 1992). The plasmid was linearized with NotI, and cRNA was transcribed in vitro with T3 RNA polymerase (mMESSAGE mMACHINE kit; Ambion). Microinjection into oocytes was performed as described previously (Yokosho et al., 2011). The transport activities for DMA, NA, and Fe(III)-DMA were measured using the two-electrode voltage-clamp method. Fifty nanoliters of cRNA (1 ng nL−1) was injected into the selected oocytes using a Nanoject II automatic injector (Drummond Scientific). After 1 d of incubation with Modified Barth’s Saline (MBS) solution buffer at 18°C, 10 independent OsVMT-expressed oocytes were injected with 50 mm different substrates [DMA, Fe(III)-DMA, and NA]. The same number of water-injected oocytes was used as a control. The net current was measured using the two-electrode voltage clamp system with the amplifier (MEZ-7200 and CEZ-1200; Nihon Kohden) at −100 mV clamped oocyte membrane. DMA was purified from the root exudates of Fe-deficient wheat (Triticum aestivum) according to the procedures described by Ma and Nomoto (1993). NA was purchased from T Hasegawa. Fe(III)-DMA was prepared by mixing FeCl3 with DMA at a ratio of 1:1 at room temperature for 3 h (Schaaf et al., 2004).

Furthermore, to investigate direct efflux from the oocytes, 50 nL of cRNA (1 ng nL−1) or water was injected into the selected oocytes. After a 1-d incubation in MBS solution buffer at 18°C, six to 10 oocytes per sample were injected with 50 nL of 50 mm DMA, followed by transferring into tubes. The cells were immediately washed with MBS buffer four times and then incubated in 500 μL of MBS buffer. The external buffer was collected after 1 and 3 h of incubation. Finally, the oocytes were homogenized with HPLC mobile phase buffer (0.15 m lithium citrate, pH 2.6). DMA concentrations in the external solution and oocytes were determined by HPLC as described below in "Determination of DMA and Citrate". The efflux activity was expressed as a percentage (DMA contents in external solution/total DMA content [DMA in external solution + remaining in the oocytes] × 100).

Generation of osvmt Knockout Lines by CRISPR/Cas9

The Cas9 plant expression vector (pU6gRNA) and single guide RNA expression vector (pZDgRNA_Cas9ver.2_HPT) were kindly provided by Dr. Masaki Endo (National Institute of Agrobiological Sciences, Japan; Mikami et al., 2015). According to the design principles of the target sequences in the CRISPR/Cas9 system, 20 bases upstream of the PAM motif were selected as candidate target sequences. The target sequences in the ORF region of OsVMT are listed in Supplemental Table S1. Double-stranded target sequences (made by annealing the paired single oligonucleotides at 95°C for 5 min) were cloned into pU6gRNA vector (linearized by BbsI). OsU3-gYSA in pZDgRNA-Cas9ver.2_HPT was replaced by synthetic guide RNA expression constructs using AscI and PacI sites. The derived construct was transformed into calluses (cv Nipponbare) by A. tumefaciens-mediated transformation (Hiei et al., 1994).

To genotype the resultant mutants, genomic DNA was extracted from leaves of transgenic rice plants. PCR amplifications were carried out using primer pairs flanking the designed target sites as listed in Supplemental Table S1. The PCR products (about 500 bp) were sequenced directly using internal specific primers, of which the binding positions are desirably at about 200 bp upstream of the target sites. Two homologous knockout lines without Cas9 were selected, and the T3 generation was used in the following phenotypic analysis.

Physiological Analysis of vmt Mutant

Seedlings (2 weeks old) of vmt mutants and the wild type prepared as described above were exposed to a nutrient solution containing different FeSO4 concentrations (0, 0.2, 2, or 10 μm) or different Zn concentrations (0.04, 0.4, or 4 μm). The solution was renewed every 2 d. After a 2-week exposure, the roots, shoot basal region, and shoots were sampled for growth analysis and mineral element determination as described below in "Metal Concentration Determination".

For soil culture, 13-d-old seedlings grown hydroponically were transplanted to a 3.5-L plastic pot filled with paddy soil. Tap water was supplied daily. At the ripening stage, the plant was harvested and the yield was recorded. After air drying, the spikelet number per panicle was counted. The percentage of filled spikelets was determined in a salt solution with a gravity of 1.06. Furthermore, the plants were separated into brown rice, husk, rachis, peduncle, nodes I and II, flag leaf blade, flag leaf sheath, and straw (containing the internodes and leaf II to leaf VI). The polished grain was prepared by a grain polisher (Kett grain polisher Pearlest; Kett Electric Laboratory). The concentration of mineral elements was determined in each organ as described below in "Metal Concentration Determination".

Isolation of Apoplastic and Symplastic Solutions

Dehusked seeds of both wild-type rice and vmt mutants were soaked in water overnight at 30°C in the dark and then transferred to a net floating on a 0.5 mm CaCl2 solution. After 2 d, the seedlings were exposed to a 0.5 mm CaCl2 solution containing 2 μm FeSO4 for 1 d. Apoplastic and symplastic solutions were obtained by centrifugation from the root tips (0–1 cm) and basal roots (1–2 cm) according to the methods of Ma et al. (2004) with slight modifications. For each sample, 20 roots were first washed with a cold 5 mm CaCl2 solution three times, and then root segments (0–1 and 1–2 cm) were excised with a razor blade and blotted dry. The root segments were placed in a 0.45-μm filter unit (Ultrafree; Merck Millipore) with the cut ends facing down and centrifuged at 2,000g for 15 min at 4°C to obtain the apoplastic solution. After centrifugation, root segments were frozen at −80°C and then thawed at room temperature. The symplastic solution was prepared from the frozen-thawed tissues by centrifuging at 20,600g for 15 min at 4°C. The Fe concentration in the apoplastic and symplastic solutions was determined by inductively coupled plasma mass spectrometry as described below in "Metal Concentration Determination".

Analysis of Xylem Sap and Root Cell Sap

To compare the concentrations of Fe, Zn, DMA, and citrate in the xylem sap or root cell sap between the wild type and vmt mutants, seedlings (18 d old) of both wild-type rice and vmt mutants were exposed to a nutrient solution containing 2 μm FeSO4. The solution was renewed every 2 d. After 1 week, xylem sap and root cell sap were collected and subjected to determination of Fe, Zn, DMA, and citrate as described below. For collection of xylem sap, the shoot (2 cm above the root) was excised with a razor, and then the xylem sap was collected with a micropipette for 1 h after decapitation of the shoot. For collection of root cell sap, the excised roots were placed on a filter in a tube and frozen at −80°C overnight. After thawing at room temperature, the cell sap was collected by centrifugation at 20,600g for 10 min (Xia et al., 2010). The concentration of Fe3+ and Fe2+ in the xylem sap and root cell sap was determined by the QuantiChrom Fe assay kit (Bioassay System) according to the manufacturer’s instructions.

Metal Concentration Determination

All samples were dried at 70°C in an oven for 4 d. Dried samples were digested with concentrated HNO3 (60%, w/w) at a temperature up to 140°C. The metal concentrations in the digested solution, root apoplastic and symplastic solution, xylem sap, and root cell sap were determined by inductively coupled plasma mass spectrometry (7700X; Agilent Technologies).

Determination of DMA and Citrate

DMA in the xylem sap, root cell sap, oocyte, and polished grain was quantified with HPLC using a cation-exchange column (Shim-pack; Amino-Li, Shimadzu). The polished grains were crushed in a Multibeads shocker (Yasui Kikai). Approximately 100 mg of the crushed grains was mixed with 2 mL of deionized water and incubated at 100°C for 20 min with occasional vortex mixing to extract DMA. After that, the samples were centrifuged at 15,000 rpm for 20 min, and the supernatant was collected. These extraction steps were repeated three times. The combined supernatant was diluted eight times for measurement with HPLC. The mobile phase was 0.15 m lithium citrate (pH 2.6) mixed with 0.2 m LiOH at a proportion of 5% (v/v). The total flow rate of the mobile phase was 0.4 mL min−1 at 50°C. Detection of fluorescence was conducted after reaction with NaClO and o-phthalaldehyde at an emission of 450 nm and excitation of 350 nm. The concentration of DMA was calculated based on the peak area. The concentration of citrate was analyzed by an enzymatic method as described previously (Delhaize et al., 1993; Yokosho et al., 2010).

Statistical Analysis

Tukey’s test was applied to test differences among treatments at P < 0.05 using SPSS 16.0 (SPSS).

Accession Number

Sequence data from this article can be found in the GenBank/EMBL databases under accession number LC492899 (OsVMT).

Supplemental Data

The following supplemental materials are available.

Acknowledgments

We thank Dr. Masaki Endo for providing pU6gRNA and pZH_gYSA_MMCas9 for the generation of CRISPR/Cas9 lines.

Footnotes

1

This work was supported by a Grant-in-Aid for Specially Promoted Research from the Japan Society for the Promotion of Science (16H06296 to J.F.M.).

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References

  1. Ariga T, Hazama K, Yanagisawa S, Yoneyama T (2014) Chemical forms of iron in xylem sap from graminaceous and non-graminaceous plants. Soil Sci Plant Nutr 60: 460–469 [Google Scholar]
  2. Bashir K, Ishimaru Y, Nishizawa NK (2012) Molecular mechanisms of zinc uptake and translocation in rice. Plant Soil 361: 189–201 [Google Scholar]
  3. Bouis HE, Hotz C, McClafferty B, Meenakshi JV, Pfeiffer WH (2011) Biofortification: A new tool to reduce micronutrient malnutrition. Food Nutr Bull (Suppl) 32: S31–S40 [DOI] [PubMed] [Google Scholar]
  4. Bughio N, Yamaguchi H, Nishizawa NK, Nakanishi H, Mori S (2002) Cloning an iron-regulated metal transporter from rice. J Exp Bot 53: 1677–1682 [DOI] [PubMed] [Google Scholar]
  5. Cai H, Huang S, Che J, Yamaji N, Ma JF (2019) The tonoplast-localized transporter OsHMA3 plays an important role in maintaining Zn homeostasis in rice. J Exp Bot 70: 2717–2725 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen S, Tao L, Zeng L, Vega-Sanchez ME, Umemura K, Wang GL (2006) A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice. Mol Plant Pathol 7: 417–427 [DOI] [PubMed] [Google Scholar]
  7. Connolly EL, Guerinot ML (2002) Iron stress in plants. Genome Biol 3: 1024.1–1024.4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Delhaize E, Ryan PR, Randall PJ (1993) Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiol 103: 695–702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fuse T, Sasaki T, Yano M (2001) Ti-plasmid vectors useful for functional analysis of rice genes. Plant Biotechnol 18: 219–222 [Google Scholar]
  10. Gernand AD, Schulze KJ, Stewart CP, West KP Jr, Christian P (2016) Micronutrient deficiencies in pregnancy worldwide: Health effects and prevention. Nat Rev Endocrinol 12: 274–289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Haydon MJ, Cobbett CS (2007) A novel major facilitator superfamily protein at the tonoplast influences zinc tolerance and accumulation in Arabidopsis. Plant Physiol 143: 1705–1719 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Haydon MJ, Kawachi M, Wirtz M, Hillmer S, Hell R, Krämer U (2012) Vacuolar nicotianamine has critical and distinct roles under iron deficiency and for zinc sequestration in Arabidopsis. Plant Cell 24: 724–737 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282 [DOI] [PubMed] [Google Scholar]
  14. Higuchi K, Watanabe S, Takahashi M, Kawasaki S, Nakanishi H, Nishizawa NK, Mori S (2001) Nicotianamine synthase gene expression differs in barley and rice under Fe-deficient conditions. Plant J 25: 159–167 [DOI] [PubMed] [Google Scholar]
  15. Inoue H, Kobayashi T, Nozoye T, Takahashi M, Kakei Y, Suzuki K, Nakazono M, Nakanishi H, Mori S, Nishizawa NK (2009) Rice OsYSL15 is an iron-regulated iron(III)-deoxymugineic acid transporter expressed in the roots and is essential for iron uptake in early growth of the seedlings. J Biol Chem 284: 3470–3479 [DOI] [PubMed] [Google Scholar]
  16. Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, Wada Y, Watanabe S, Matsuhashi S, Takahashi M, et al. (2006) Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. Plant J 45: 335–346 [DOI] [PubMed] [Google Scholar]
  17. Ishimaru Y, Masuda H, Bashir K, Inoue H, Tsukamoto T, Takahashi M, Nakanishi H, Aoki N, Hirose T, Ohsugi R, et al. (2010) Rice metal-nicotianamine transporter, OsYSL2, is required for the long-distance transport of iron and manganese. Plant J 62: 379–390 [DOI] [PubMed] [Google Scholar]
  18. Johnson AA, Kyriacou B, Callahan DL, Carruthers L, Stangoulis J, Lombi E, Tester M (2011) Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS ONE 6: e24476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kakei Y, Yamaguchi I, Kobayashi T, Takahashi M, Nakanishi H, Yamakawa T, Nishizawa NK (2009) A highly sensitive, quick and simple quantification method for nicotianamine and 2′-deoxymugineic acid from minimum samples using LC/ESI-TOF-MS achieves functional analysis of these components in plants. Plant Cell Physiol 50: 1988–1993 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kobayashi T, Nagasaka S, Senoura T, Itai RN, Nakanishi H, Nishizawa NK (2013) Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation. Nat Commun 4: 2792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33: 1870–1874 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lanquar V, Lelièvre F, Bolte S, Hamès C, Alcon C, Neumann D, Vansuyt G, Curie C, Schröder A, Krämer U, et al. (2005) Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron. EMBO J 24: 4041–4051 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lee S, Chiecko JC, Kim SA, Walker EL, Lee Y, Guerinot ML, An G (2009) Disruption of OsYSL15 leads to iron inefficiency in rice plants. Plant Physiol 150: 786–800 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ma JF, Nomoto K (1993) Two related biosynthetic pathways of mugineic acids in gramineous plants. Plant Physiol 102: 373–378 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ma JF, Tamai K, Ichii M, Wu GF (2002) A rice mutant defective in Si uptake. Plant Physiol 130: 2111–2117 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ma JF, Mitani N, Nagao S, Konishi S, Tamai K, Iwashita T, Yano M (2004) Characterization of the silicon uptake system and molecular mapping of the silicon transporter gene in rice. Plant Physiol 136: 3284–3289 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Masuda H, Ishimaru Y, Aung MS, Kobayashi T, Kakei Y, Takahashi M, Higuchi K, Nakanishi H, Nishizawa NK (2012) Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition. Sci Rep 2: 543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mikami M, Toki S, Endo M (2015) Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol 88: 561–572 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moore KL, Chen Y, van de Meene AM, Hughes L, Liu W, Geraki T, Mosselmans F, McGrath SP, Grovenor C, Zhao FJ (2014) Combined NanoSIMS and synchrotron X-ray fluorescence reveal distinct cellular and subcellular distribution patterns of trace elements in rice tissues. New Phytol 201: 104–115 [DOI] [PubMed] [Google Scholar]
  30. Negishi T, Nakanishi H, Yazaki J, Kishimoto N, Fujii F, Shimbo K, Yamamoto K, Sakata K, Sasaki T, Kikuchi S, et al. (2002) cDNA microarray analysis of gene expression during Fe-deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe-deficient barley roots. Plant J 30: 83–94 [DOI] [PubMed] [Google Scholar]
  31. Nishiyama R, Kato M, Nagata S, Yanagisawa S, Yoneyama T (2012) Identification of Zn-nicotianamine and Fe-2′-deoxymugineic acid in the phloem sap from rice plants (Oryza sativa L.). Plant Cell Physiol 53: 381–390 [DOI] [PubMed] [Google Scholar]
  32. Nishizawa N, Mori S (1987) The particular vesicle appearing in barley root-cells and its relation to mugineic acid-secretion. J Plant Nutr 10: 1013–1020 [Google Scholar]
  33. Nozoye T, Nagasaka S, Kobayashi T, Takahashi M, Sato Y, Sato Y, Uozumi N, Nakanishi H, Nishizawa NK (2011) Phytosiderophore efflux transporters are crucial for iron acquisition in graminaceous plants. J Biol Chem 286: 5446–5454 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nozoye T, Nagasaka S, Bashir K, Takahashi M, Kobayashi T, Nakanishi H, Nishizawa NK (2014) Nicotianamine synthase 2 localizes to the vesicles of iron-deficient rice roots, and its mutation in the YXXφ or LL motif causes the disruption of vesicle formation or movement in rice. Plant J 77: 246–260 [DOI] [PubMed] [Google Scholar]
  35. Nozoye T, Nagasaka S, Kobayashi T, Sato Y, Uozumi N, Nakanishi H, Nishizawa NK (2015) The phytosiderophore efflux transporter TOM2 is involved in metal transport in rice. J Biol Chem 290: 27688–27699 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Preston GM, Carroll TP, Guggino WB, Agre P (1992) Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385–387 [DOI] [PubMed] [Google Scholar]
  37. Ramesh SA, Shin R, Eide DJ, Schachtman DP (2003) Differential metal selectivity and gene expression of two zinc transporters from rice. Plant Physiol 133: 126–134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ricachenevsky FK, Sperotto RA, Menguer PK, Sperb ER, Lopes KL, Fett JP (2011) ZINC-INDUCED FACILITATOR-LIKE family in plants: Lineage-specific expansion in monocotyledons and conserved genomic and expression features among rice (Oryza sativa) paralogs. BMC Plant Biol 11: 20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sasaki A, Yamaji N, Mitani-Ueno N, Kashino M, Ma JF (2015) A node-localized transporter OsZIP3 is responsible for the preferential distribution of Zn to developing tissues in rice. Plant J 84: 374–384 [DOI] [PubMed] [Google Scholar]
  40. Schaaf G, Ludewig U, Erenoglu BE, Mori S, Kitahara T, von Wirén N (2004) ZmYS1 functions as a proton-coupled symporter for phytosiderophore- and nicotianamine-chelated metals. J Biol Chem 279: 9091–9096 [DOI] [PubMed] [Google Scholar]
  41. Senoura T, Sakashita E, Kobayashi T, Takahashi M, Aung MS, Masuda H, Nakanishi H, Nishizawa NK (2017) The iron-chelate transporter OsYSL9 plays a role in iron distribution in developing rice grains. Plant Mol Biol 95: 375–387 [DOI] [PubMed] [Google Scholar]
  42. Song WY, Yamaki T, Yamaji N, Ko D, Jung KH, Fujii-Kashino M, An G, Martinoia E, Lee Y, Ma JF (2014) A rice ABC transporter, OsABCC1, reduces arsenic accumulation in the grain. Proc Natl Acad Sci USA 111: 15699–15704 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Suzuki M, Tsukamoto T, Inoue H, Watanabe S, Matsuhashi S, Takahashi M, Nakanishi H, Mori S, Nishizawa NK (2008) Deoxymugineic acid increases Zn translocation in Zn-deficient rice plants. Plant Mol Biol 66: 609–617 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Trijatmiko KR, Dueñas C, Tsakirpaloglou N, Torrizo L, Arines FM, Adeva C, Balindong J, Oliva N, Sapasap MV, Borrero J, et al. (2016) Biofortified indica rice attains iron and zinc nutrition dietary targets in the field. Sci Rep 6: 19792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Ueno D, Yamaji N, Kono I, Huang CF, Ando T, Yano M, Ma JF (2010) Gene limiting cadmium accumulation in rice. Proc Natl Acad Sci USA 107: 16500–16505 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Xia J, Yamaji N, Kasai T, Ma JF (2010) Plasma membrane-localized transporter for aluminum in rice. Proc Natl Acad Sci USA 107: 18381–18385 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yamaguchi N, Ishikawa S, Abe T, Baba K, Arao T, Terada Y (2012) Role of the node in controlling traffic of cadmium, zinc, and manganese in rice. J Exp Bot 63: 2729–2737 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yamaji N, Ma JF (2007) Spatial distribution and temporal variation of the rice silicon transporter Lsi1. Plant Physiol 143: 1306–1313 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yamaji N, Ma JF (2014) The node, a hub for mineral nutrient distribution in graminaceous plants. Trends Plant Sci 19: 556–563 [DOI] [PubMed] [Google Scholar]
  50. Yamaji N, Ma JF (2017) Node-controlled allocation of mineral elements in Poaceae. Curr Opin Plant Biol 39: 18–24 [DOI] [PubMed] [Google Scholar]
  51. Yamaji N, Ma JF (2019) Bioimaging of multiple elements by high-resolution LA-ICP-MS reveals altered distribution of mineral elements in the nodes of rice mutants. Plant J doi:10.1111/tpj.14410 [DOI] [PubMed] [Google Scholar]
  52. Yamaji N, Sasaki A, Xia JX, Yokosho K, Ma JF (2013a) A node-based switch for preferential distribution of manganese in rice. Nat Commun 4: 2442. [DOI] [PubMed] [Google Scholar]
  53. Yamaji N, Xia J, Mitani-Ueno N, Yokosho K, Ma JF (2013b) Preferential delivery of zinc to developing tissues in rice is mediated by P-type heavy metal ATPase OsHMA2. Plant Physiol 162: 927–939 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yang X, Ye ZQ, Shi CH, Zhu ML, Graham RD (1998) Genotypic differences in concentrations of iron, manganese, copper, and zinc in polished rice grains. J Plant Nutr 21: 1453–1462 [Google Scholar]
  55. Yokosho K, Yamaji N, Ueno D, Mitani N, Ma JF (2009) OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice. Plant Physiol 149: 297–305 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yokosho K, Yamaji N, Ma JF (2010) Isolation and characterisation of two MATE genes in rye. Funct Plant Biol 37: 296–303 [Google Scholar]
  57. Yokosho K, Yamaji N, Ma JF (2011) An Al-inducible MATE gene is involved in external detoxification of Al in rice. Plant J 68: 1061–1069 [DOI] [PubMed] [Google Scholar]
  58. Yokosho K, Yamaji N, Ma JF (2016a) OsFRDL1 expressed in nodes is required for distribution of iron to grains in rice. J Exp Bot 67: 5485–5494 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Yokosho K, Yamaji N, Mitani-Ueno N, Shen RF, Ma JF (2016b) An aluminum-inducible IREG gene is required for internal detoxification of aluminum in buckwheat. Plant Cell Physiol 57: 1169–1178 [DOI] [PubMed] [Google Scholar]
  60. Zhang Y, Xu YH, Yi HY, Gong JM (2012) Vacuolar membrane transporters OsVIT1 and OsVIT2 modulate iron translocation between flag leaves and seeds in rice. Plant J 72: 400–410 [DOI] [PubMed] [Google Scholar]
  61. Zhao FJ, Moore KL, Lombi E, Zhu YG (2014) Imaging element distribution and speciation in plant cells. Trends Plant Sci 19: 183–192 [DOI] [PubMed] [Google Scholar]

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