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First published online October 19, 2007; 10.1104/pp.107.107912 Plant Physiology 145:1647-1657 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Mutation in Nicotianamine Aminotransferase Stimulated the Fe(II) Acquisition System and Led to Iron Accumulation in Rice1,[C],[W],[OA]The State Key Laboratory of Plant Physiology and Biochemistry, College of Life Science, Zhejiang University, Hangzhou 310058, China (L.C., F.W., H.S., F. Huang, L.Z., F. He, J.L., P.W.); Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom (F.-J.Z.); and Research Institute for Bioresources, Okayama University, Kurashiki 710–0046, Japan (D.U., J.-F.M.)
Higher plants acquire iron (Fe) from the rhizosphere through two strategies. Strategy II, employed by graminaceous plants, involves secretion of phytosiderophores (e.g. deoxymugineic acid in rice [Oryza sativa]) by roots to solubilize Fe(III) in soil. In addition to taking up Fe in the form of Fe(III)-phytosiderophore, rice also possesses the strategy I-like system that may absorb Fe(II) directly. Through mutant screening, we isolated a rice mutant that could not grow with Fe(III)-citrate as the sole Fe source, but was able to grow when Fe(II)-EDTA was supplied. Surprisingly, the mutant accumulated more Fe and other divalent metals in roots and shoots than the wild type when both were supplied with EDTA-Fe(II) or grown under water-logged field conditions. Furthermore, the mutant had a significantly higher concentration of Fe in both unpolished and polished grains than the wild type. Using the map-based cloning method, we identified a point mutation in a gene encoding nicotianamine aminotransferase (NAAT1), which was responsible for the mutant phenotype. Because of the loss of function of NAAT1, the mutant failed to produce deoxymugineic acid and could not absorb Fe(III) efficiently. In contrast, nicotianamine, the substrate for NAAT1, accumulated markedly in roots and shoots of the mutant. Microarray analysis showed that the expression of a number of the genes involved in Fe(II) acquisition was greatly stimulated in the naat1 mutant. Our results demonstrate that disruption of deoxymugineic acid biosynthesis can stimulate Fe(II) acquisition and increase iron accumulation in rice.
Iron (Fe) is an important mineral for both plant production and human nutrition. Fe deficiency is one of the most prevalent nutrient deficiencies in the world, affecting an estimated 2 billion people, especially in the area where vegetable-based diets are the primary food source (World Health Organization, 2002  ). Rice (Oryza sativa), the dominant cereal crop in many developing countries, particularly contains low Fe in its polished grains (Barry, 2006). To alleviate the problem of severe Fe deficiency worldwide, an international Fe biofortification program has been organized by the HarvestPlus global initiative (http://www.harvestplus.org) to develop Fe-rich rice varieties using both traditional breeding and biotechnological approaches. Most efforts have been directed toward screening for high Fe content materials from various sources of germplasm. To use biotechnological methods for Fe biofortification requires that the molecular mechanisms controlling Fe acquisition in rice be understood.
In aerobic soils, Fe is present mainly as Fe(III) oxides, which have very low solubility and are not readily available to plants (Guerinot and Yi, 1994
Fe acquisition by strategy II plants is characterized by secretion of Fe(III)-chelating substances (phytosiderophores) and uptake of intact Fe(III)-phytosiderophore complexes (Takagi et al., 1984
NA is not only a key intermediate for the biosynthesis of MA for strategy II plants, but also an important metal chelator that can facilitate transport of Fe and other transition metals inside plants (von Wiren et al., 1999
Recently, it has been shown that rice possesses not only a strategy II but also a strategy I-like system that may take up Fe(II) via Fe(II) transporters OsIRT1 and OsIRT2 (Ishimaru et al., 2006 In this study, we isolated and characterized a rice mutant whose strategy II system was interrupted due to the loss of a functional NAAT enzyme. Surprisingly, the mutant exhibited significant elevation of Fe and other metal concentrations in both seedlings and seeds when plants were supplied with Fe(II). Physiological and molecular analysis showed that diminishing MA synthesis in the naat1 mutant stimulated Fe(II) acquisition system and led to Fe accumulation. Our results demonstrate the importance of the Fe(II) uptake strategy in rice Fe acquisition.
Isolation and Gene Cloning of the naat1 Mutant
Seeds from the M2 generation of an ethyl methane sulfonate-mutagenized population of rice ‘Nipponbare’ were germinated and grown in a nutrient solution [containing Fe(III); Yoshida et al., 1976
With the same nutrient solution, the mutant was unable to survive longer than 20 d after germination. To rescue the mutant, we changed various components in the nutrient solution. When citrate-Fe(III) was replaced by EDTA-Fe(II), growth of roots and shoots of mutant plants was normal and chlorophyll content (SPAD value) of the mutant seedlings was at the same level as that of wild-type seedlings (Fig. 1, A and G; Supplemental Table S1). Similarly, normal growth was observed when FeSO4 was used in the nutrient solution, whereas FeCl3 could not restore normal growth (data not shown). These results suggested that the mutant could utilize Fe(II), but not Fe(III).
Genetic analysis showed that a single recessive gene was responsible for the mutant phenotype. Using 2,900 F2 mutant seedlings selected from a F2 population derived from a cross between the mutant and the Indica cultivar ‘Kasalath’, the mutation was mapped to a 123-kb region between RM13046 and RM13051 (Fig. 2A
). This region contains 18 open reading frames (ORFs), including the NICOTIANAMINE AMINOTRANSFERASE1 (NAAT1) gene (LOC_Os02g20360.1; Inoue et al., 2004
Confirmation that the point mutation in naat1 was responsible for the mutant phenotype was achieved by genetic complementation. The wild-type full-length ORF of NAAT1 was inserted into the binary vector pTF101.1 under the control of a maize ubiquitin-1 (Ubi-1) promoter and a nopaline synthase terminator (Frame et al., 2002 ). The resulting construct was used to introduce the complete NAAT1 gene into the genome of the naat1 mutant via Agrobacterium-mediated transformation (Chen et al., 2003). Transgenic mutant lines expressing both the shortened and the full-length NAAT1 mRNA displayed normal growth phenotype when grown in the presence of citrate-Fe(III) (Fig. 1D).
To investigate whether disruption of the NAAT1 enzyme affects accumulation of the NAAT1 substrate NA, we measured NA content in shoots and roots of 10-d-old naat1 and wild-type seedlings with a supply of 35 µM citrate-Fe(III), 125 µM EDTA-Fe(II), or no Fe. Results showed that both shoots and roots of wild-type plants contained a low concentration of NA, ranging from 0.038 to 0.088 µg g–1 fresh weight in all treatments (Fig. 3B ). In contrast, NA concentrations in naat1 shoots and roots were greatly elevated compared with those of the wild type. NA concentrations of the mutant were 3- to 43-fold higher than those of the wild type (Fig. 3B).
Biosynthesis of DMA Is Blocked in naat1 Plants
NAAT is a critical enzyme in the biosynthesis of MA. It catalyzes the transfer of the amino group from NA to form the precursor of DMA, the 3''-oxo form of DMA. To determine the secretion of DMA, seedlings of the wild type and naat1 were precultured with one-half-strength Kimura nutrient solution containing 125 µM Fe(II)-EDTA (pH 5.5; Ma et al., 2001 Results showed that, without Fe(II) supply, both wild-type and naat1 mutant plants developed typical Fe-deficient chlorotic symptoms in the treatment, but were normal in the Fe(II) supply condition (Fig. 3A). Root exudates were collected and measured on day 13 after treatment. Wild-type roots secreted considerable amounts of DMA: 151 and 223 nmol g–1 dry weight in Fe(II) or no-Fe supply treatments, respectively (Fig. 3C). In contrast, naat1 roots secreted negligible amounts of DMA in both Fe(II) or no-Fe treatments, indicating that the loss of function of NAAT1 blocked the production and secretion of DMA. For comparison, wild-type roots supplied with 125 µM Fe(III)-EDTA secreted little DMA (data not shown).
To evaluate the effect of disruption of the NAAT1 enzyme on the rice Fe absorption system, we performed Affymetrix GeneChip analysis on the leaves and roots of the 10-d-old wild-type and naat1 seedlings grown with either 35 µM citrate-Fe(III), 125 µM EDTA-Fe(II), or no Fe. Gene expression patterns from the microarray analysis were verified by RT-PCR for 12 genes encoding Fe uptake or transport proteins. The results from RT-PCR analysis were consistent with those from the microarray analysis (Table I ; Fig. 4 ).
Table I shows that a number of genes involved in various steps of Fe(II) uptake and transport were up-regulated in the naat1 mutant seedlings under no-Fe, Fe(III), or Fe(II) supply conditions. When Fe(III) was supplied as the sole Fe source, all Fe deficiency-inducible genes examined were up-regulated in roots and/or leaves of the naat1 mutant plants compared with the wild-type counterparts (Table I), which were consistent with an Fe deficiency response (Inoue et al., 2003 ; Kobayashi et al., 2005). These results confirmed that the naat1 mutant was not able to utilize Fe(III).
Because the naat1 mutant can only absorb Fe in the form of Fe(II), the difference in the gene expression pattern between wild-type and naat1 plants under the Fe(II) supply conditions should reflect their difference in the Fe(II) uptake capacity. Microarray data showed that a group of genes involved in Fe(II) uptake and transport were greatly activated in naat1 seedlings under Fe(II) supply conditions. OsNRAMP1 belongs to the same family as AtNRAMP1 and GmNRAMP1, which function as ferrous transporters in Arabidopsis and soybean (Glycine max; Curie et al., 2000
Significant induction of OsIRO2, which binds preferentially to sequences upstream of Fe deficiency-inducible genes, such as OsNAS1, OsNAS3, and OsIRT1 (Ogo et al., 2006 Under Fe(II) supply conditions, expression of OsNAS1, OsNAS2, and OsNAS3 in the naat1 roots was 3.9, 3.5, and 4.1 times higher than those in wild-type plants. Induction of NAS genes may have contributed to the significant accumulation of NA in naat1 shoots and roots, in addition to the markedly reduced consumption of NA for MA biosynthesis in the mutant (Fig. 3B).
We measured the uptake and translocation of Fe and other metal elements in wild-type and naat1 plants harvested from the DMA secretion experiment. In the treatment with Fe(II)-EDTA, Fe concentrations in the naat1 shoots and roots were 15.8% and 41.8% higher than those in wild-type seedlings (Fig. 3D), indicating that the naat1 mutant had enhanced ferrous uptake system. These results were further supported by the finding that the naat1 mutant contained significantly higher (58%–92%) concentrations of Zn in shoots and roots than the wild type (Fig. 3E). Furthermore, when 1 µM cadmium (Cd) was added to the nutrient solution, Cd concentrations in both naat1 roots or shoots were about 50% higher than that those in wild-type seedlings (Fig. 3F). The naat1 mutant grew normally in paddy soil and accumulated more Fe in seeds than wild-type plants in a field experiment. Despite the fact that the naat1 mutant was unable to absorb Fe(III) through strategy II, plants grew normally in water-logged paddy soils where ferrous iron was abundant (Fig. 1C). Agronomic traits, including the duration of maturation, plant height, tiller number, seed set rate, seed number per panicle, and seed weight of naat1 and wild-type plants, were evaluated in a field experiment. Growing conditions used in the field experiment were the same as those for normal paddy rice, for which continuous flooding water was provided until the late grain-filling stage. No significant (P > 0.05) differences between mutant and wild-type plants were observed in all agronomic traits evaluated (Table II ). Elemental analysis showed that the naat1 straw contained 2.7, 3.9, and 1.8 times more Fe, Zn, and Cd, respectively, than wild-type straw (Fig. 5A ). Fe concentrations in the naat1 unpolished and polished grains were 24.7 and 4.6 mg kg–1 dry weight, respectively, which were 1.8 and 3.8 times higher than their wild-type counterparts. Although naat1 plants contained more Zn and Cd in the seedlings and straws (Fig. 3, D–F), Zn concentrations in the unpolished and polished grains remained the same as in the wild-type grain, and the Cd concentrations were lower than those of the wild-type grain (Fig. 5B). Accumulation of other divalent metals, including copper (Cu) and Mn, showed a similar pattern as for Zn and Cd between naat1 and wild-type plants (Supplemental Table S2).
MA is a natural Fe chelator secreted from roots of graminaceous plants that solubilizes Fe in the soil. Biosynthesis and secretion of MA are therefore crucial to the acquisition of Fe(III) in strategy II plants. NAAT is a critical enzyme in the biosynthesis of MA from L-Met. BLAST searches identified five other putative NAAT genes in rice, named OsNAAT-L1 to OsNAAT-L5. Phylogenetic analysis indicated that NAAT1 is closely clustered with barley NAAT-A and NAAT-B (Fig. 2D; Takahashi et al., 1999 ). Our microarray and RT-PCR analysis showed that only OsNAAT1, among all NAAT-like genes analyzed, was induced by Fe deficiency (data not shown). We also observed that naat1 mutant plants lost the ability to synthesize and secrete DMA regardless of the status of Fe supply (Fig. 3A). Taken together, we conclude that, among the six rice NAAT-like genes, only OsNAAT1 is involved in the production of DMA. Due to the loss of function of OsNAAT1, the naat1 mutant could not survive in aerobic soils or in hydroponic cultures with Fe(III) as the sole Fe source.
However, Fe(II) is abundant in flooded paddy soils. Rice can utilize Fe(II) directly through the strategy I-like system, even though it has no functional ferric reductase to reduce Fe(III) to Fe(II) (Ishimaru et al., 2006
In the naat1 mutant, although the greatly diminished DMA secretion blocked the absorption of Fe(III) through the strategy II system, activation of the Fe(II) uptake system appears to overcompensate for the loss of strategy II, resulting in enhanced uptake of Fe when Fe(II) was available. Our results concur with the findings of Ishimaru et al. (2006 In naat1 rice, the Fe(II) uptake system was enhanced, resulting in increased accumulation of Fe, Zn, Cd, and other divalent metals in the mutant roots and shoots in the hydroponic experiments and in the mature straw from the field experiment (Fig. 5, A–C). However, only Fe concentration was significantly enhanced in the grain. It is likely that there is one (or more) possible Fe-specific transporter that transports the accumulated Fe from shoots to grains. Further investigations are needed to identify any Fe-specific transporters involved in the translocation of Fe from shoot tissues to grains.
Data from the HarvestPlus program show that commercial varieties of rice normally contain about 2 mg kg–1 Fe and 12 mg kg–1 Zn in the polished grain (Barry, 2006
Plant Materials and Growth Conditions
The rice (Oryza sativa) mutant naat1 was identified in an ethyl methane sulfonate-mutagenized population from the cultivar ‘Nipponbare’ in culture solution prepared as described by Yoshida et al. (1976)
For NA analysis, Affymetrix GeneChip assay, and SPAD measurement, naat1 mutant and wild-type seeds were germinated in distilled water for 2 d. After germination, 15 seedlings were transferred to a plastic net floating on the Yoshida nutrient solution (Yoshida et al., 1976
Seedlings used for measuring metal uptake and DMA secretion were prepared as follows. The naat1 and wild-type seeds were germinated in tap water and placed on a plastic net floating on 0.5 mM CaSO4 solution for 10 d, then transferred to 1.2-L pots (three seedlings/pot) containing one-half-strength Kimura B solution (Ma et al., 2001 A field experiment was carried out to compare the growth performance and metal uptake of the naat1 mutant and the wild type. Pregerminated seeds of naat1 and wild type were planted in a paddy field on the farm of the Huajia campus, Zhejiang University, with a planting distance of 18 x 18 cm. The soil contained 10.92 g kg–1 total Fe and 87.8 mg kg–1 total Zn, respectively. During the whole growing period, the paddy field was maintained with 5- to 15-cm standing water until the plants reached the late grain-filling stage. Fertilizers (nitrogen, phosphorus, and potassium) were applied at normal rates for paddy rice in the region. At the maturation stage, 10 plants each were harvested for the measurement of the agronomic traits. Dried straws and seeds were used for analysis of metal concentrations.
The NAAT1 gene was mapped to the short arm of chromosome 2 between simple sequence repeat markers RM301 and RM324 using 81 F2 mutant plants. The locus was further placed within a 123-kb region between RM13046 and RM13051 markers using 2,900 F2 mutant plants and three newly developed simple sequence repeat markers. Based on the phenotype of the mutant, the NAAT1 gene was selected out of the 18 putative proteins coded by the 123-kb DNA region as a candidate gene. Genomic DNAs and cDNAs of the gene were amplified by PCR or RT-PCR from naat1 mutant and wild-type plants. NAAT1 gene-specific forward and reverse primers are 5'-TGTCCCACACCCGTAGAAT-3' and 5'-CCATTTTGGTGACAACAGG-3'. PCR and RT-PCR products were cloned into pMD18-T vector (TaKaRa) and sequenced.
NAAT1 cDNA synthesized from the wild-type and naat1 RNA samples were cloned into the expression vector pET-29b (Novagen). The resultant plasmids were expressed using Escherichia coli strain BL21(DE3). Fresh BL21(DE3)-transformed cells were inoculated into 10 mL of Luria-Bertani/ampicillin medium and grown at 37°C until OD600 ranged from 0.7 to 0.8. Expression of NAAT1 proteins was induced by adding isopropyl thio-D-galactoside at a final concentration of 0.5 mM for an additional 4 h at 37°C. Cells were then harvested and lysed by sonication. Soluble and insoluble fractions were fractionated by centrifugation and separated by electrophoresis on 12.5% SDS-PAGE gel.
The wild-type full-length ORF of NAAT1 was amplified by RT-PCR and inserted into the binary vector pTF101.1 (Frame et al., 2002
SPAD values (total chlorophyll content) were determined on the fully expanded youngest leaves of 10-d-old seedlings with a portable chlorophyll meter (SPAD-502; Minolta Sensing).
Shoots and roots of 10-d-old naat1 and wild-type seedlings (as described in the section on plant materials and growth conditions) were harvested for NA measurement. Extraction and quantification of NA were performed as described by Weber et al. (2004)
Root exudates were collected on day 13 after –Fe(II) or +Fe(II) treatments by immersing roots in deionized water for 5 h from 8 AM to 1 PM. Solutions were passed through a cation exchange resin and eluted using 2 M NH4OH. The eluates were concentrated in a rotary evaporator at 40°C. Phytosiderophores in the root exudates were quantified with HPLC using a cation exchange column (Shim-pack; Amino-Li, Shimadzu). The mobile phase was a 0.15 M lithium citrate (pH 2.6), mixed with 0.2 M LiOH at a proportion of 5%. 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 emission of 450 nm and excitation of 350 nm. Only DMA was detected in the rice root exudates. The concentration was calculated based on the peak area.
To determine the concentrations of Fe, Zn, and Cd of the naat1 and wild-type plants, we performed elemental analysis on 38-d-old seedlings from the DMA secretion experiment and the straw and grain samples from the field experiment. In the DMA secretion experiment, plant roots and shoots were washed three times with deionized water after the collection of root exudates and dried at 70°C for 2 d. Grain samples from the field experiment were dehusked into unpolished grain. Portions of the unpolished grain samples were processed with a rice-milling machine (JNMJ3; Taizhou) for 1 min, three times, to obtain polished grain. Shoot, root, and straw samples were ground to fine powders and digested with 5 mL of 11 M HNO3 for 5 h at 150°C and metal concentrations measured by atomic absorption spectrometry (Z-2000; Hitachi). Grain samples were ground to powders first, then digested with ultrapure HNO3 and H2O2 in Teflon-coated microwave vessels, and metal concentrations measured using inductively coupled plasma mass spectrometry (Agilent 7500ce).
Ten-day-old seedlings grown in solution culture with citrate-FeCl3, EDTA-FeSO4, or no Fe were used for RNA sampling (as described in the section on plant materials and growth conditions). RNA samples from three biological replications were prepared according to the procedure recommended by the manufacturer (Affymetrix, 2003
The following materials are available in the online version of this article.
We thank Dr. Stanton Gelvin and Dr. Kan Wang for critical reading of the manuscript. We also thank Mr. Yunrong Wu for field experiment management and Dr. Ming Chen for the annotation of microarray data. Received August 22, 2007; accepted October 15, 2007; published October 19, 2007.
1 This work was supported by the National Key Basic Research Special Foundation of China (grant no. 2005CB20900), the National Natural Science Foundation (grant nos. 30471118 and 30770191), and the HarvestPlus Program China. Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council (UK). 
2 These authors contributed equally to the article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ping Wu (clspwu{at}zju.edu.cn).
[C] Some figures in this article are displayed in color online but in black and white in the print edition.
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.107912 * Corresponding author; e-mail clspwu{at}zju.edu.cn.
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ABSTRACT
RESULTS
