Cloning an iron-regulated metal transporter from rice

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1 Journal of Experimental Botany, Vol. 53, No. 374, pp. 1677±1682, July 2002 DOI: /jxb/erf004 Cloning an iron-regulated metal transporter from rice Naimatullah Bughio 1, Hirotaka Yamaguchi 1,3, Naoko K. Nishizawa 2,3, Hiromi Nakanishi 1 and Satoshi Mori 1,3,4 1 Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo, Japan 2 Laboratory of Plant Biotechnology, Department of Global Agricultural Sciences, The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo, Japan 3 Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Honcho, Kawaguchi-shi, , Saitama, Japan Received 21 September 2001; Accepted 20 March 2002 Abstract Rice cdna and genomic libraries were screened in order to clone an Fe(II) transporter gene. A cdna clone highly homologous to the Arabidopsis Fe(II) transporter gene IRT1 was isolated from Fe-de cient rice roots. The cdna clone was named OsIRT1. A genomic clone corresponding to the cdna was also obtained, sequenced and analysed. When expressed in yeast cells, OsIRT1 cdna reversed the growth defects of the yeast iron-uptake mutant. Northern blot analysis revealed that OsIRT1 mrna was predominantly expressed in roots and was induced by Fe- and Cu-de ciency. This suggests that OsIRT1 is a functional metal transporter for iron, and is actively engaged in Fe uptake from soils, especially under limiting conditions. Key words: Copper, Fe(II) transporter, iron, IRT1, rice. Introduction Decades of research on plants have established that there are two distinct iron-uptake systems based on the response of plants to Fe-de ciency. These are known as Strategy I and Strategy II (RoÈmheld, 1987; RoÈmheld and Marschner, 1986). Strategy I plants include all dicots and nongraminaceous monocots. These plants respond to Fede ciency by decreasing rhizosphere ph and reducing sparingly soluble ferric iron. These mechanisms employ releasing protons and reductants, and morphological changes in the roots. The most important step of the mechanism is the reduction of ferric iron at the root surface by membrane-resident ferric chelate reductase (Chaney et al., 1972). In Arabidopsis, this reductase is encoded by FRO2 (Robinson et al., 1999). Reduced ferrous iron is absorbed into root cells by the Fe(II) transporter. IRT1 in Arabidopsis, a member of the ZIP metal transporter family, was the rst metal transporter involved in Fe(II) transport to be identi ed in higher plants (Eide et al., 1996). IRT1 was also the rst member of the ZRT, IRT-like protein (ZIP) family to be described (Guerinot, 2000). To date, IRT1-like Fe(II) transporters have been isolated from several dicotyledonous species (Eckhardt et al., 2001; Vert et al., 2001). Strategy II iron-uptake systems are limited to graminaceous monocots. These plants release mugineic acid-family phytosiderophores to the rhizosphere, where they solubilize sparingly soluble iron by chelation. The chelated complex is then absorbed into the roots. Genes for the synthesis of phytosiderophores have been isolated from barley (Higuchi et al., 1999; Kobayashi et al., 2001; Okumura et al., 1994; Takahashi et al., 1999). The rst step of phytosiderophore synthesis is also crucial in Strategy I plants (Ling et al., 1999), which is conversion of three molecules of S-adenosyl methionine to nicotianamine. Recently, a strong candidate for the transporter of the iron±phytosiderophore complex was cloned from maize (Curie et al., 2001). It is generally believed that plants depend on a single system (either Strategy I or II) for iron acquisition. In graminaceous plants, such as barley and maize, the inducible Fe-de ciency Fe(II) transport system is either absent or is expressed only at very low levels (Zaharieva 4 To whom correspondence should be addressed. Fax: asmori@mail.ecc.u-tokyo.ac.jp ã Society for Experimental Biology 2002

2 1678 Bughio et al. and RoÈmheld, 2001). It is possible that some plants have both uptake systems. Rice is a strategy II plant and releases phytosiderophores under Fe-de ciency. In fact, rice is the rst plant in which the root-secreted iron-solubilizing substance, the phytosiderophore, was observed (Takagi, 1976). Under submerged conditions, such as in a paddy eld where rice is normally grown, however, ferrous iron is more abundant than ferric iron. Up until now, quite limited information has been available as to the existence of an Fe(II) transport system for iron uptake in rice (Eide et al., 1996). This paper reports the isolation of cdna and a genomic clone for a functional Fe(II) transporter from rice. This is the rst report of the isolation of a functional Fe(II) transporter from a Strategy II plant. It is proposed that the Fe(II) transport system participates in iron uptake in rice in the reductive environment in which rice is normally grown. Materials and methods Plant material Two varieties of rice plants, Oryza sativa cv. Notohikari or Nihonbare, were used for cdna library construction or Northern analysis, respectively. Seeds were germinated and seedlings were hydroponically grown as previously described (Mori and Nishizawa, 1987). For micronutrient de ciency treatment, 3-week-old plants were transferred to nutrient solution without Cu, Fe, Mn, or Zn, and grown for three more weeks. cdna cloning The OsIRT1 cdna clone, identical to a rice expression sequence tag (EST) (accession no. D49213) homologous to IRT1, was isolated as follows. RNA was extracted from the roots of rice plants grown under Fe-de cient conditions for 2 weeks. A cdna library was constructed according to the SUPER SCRIPT Plasmid System for cdna Synthesis and Plasmid Cloning instruction manual (Gibco- BRL, USA). The OsIRT1 cdna clone was isolated using a PCRbased strategy. Approximately 10 5 independent colonies of Escherichia coli from the library were divided into 96 sublibraries. Ninety-six pools of plasmid were prepared, and each sublibrary was screened for the EST clone by PCR using speci c primers (5 -CGAGCTGCAGGATCCGAATTCTCGGCGGCTGCATCCTGC and 5 -GGGCAACGTCCGTCTTGAAC). Any sub-library in which a fragment was ampli ed was further analysed. Repeated subdivision and screening allowed an OsIRT1 cdna clone to be isolated. Cloning a genomic sequence A commercial genomic library of rice Oryza sativa L. cv. IR36 was used for screening (CLONTECH Inc., USA) by plaque hybridization. A DNA fragment used as a probe was ampli ed using the above primer set from a cdna pool prepared from RNA isolated from 3- month-old rice leaves. DNA and RNA procedures Plasmid puri cation, subcloning, electrophoresis, blotting, and hybridization were carried out according to standard protocols (Sambrook et al., 1989). For Northern analysis, total RNA was extracted from plant materials by the SDS-phenol method (Naito et al., 1988). Total RNA (20 mg) was electrophoresed and blotted onto nylon membranes. The membranes were hybridized with 32 P- labelled probes speci c for the cdna fragments for OsIRT1. Yeast strains and growth media The following strains of the yeast, Saccharomyces cerevisiae, were used in this study: CM3260 (parent strain) MATalpha trp1-63 leu2-3,112 gcn4-101 his3-609 ura3-52, YH001 MATalpha trp1-63 leu2-3,112 gcn4-101 his3-609 ura3-52 Dftr1::URA3, YH003 MATalpha trp1-63 leu2-3,112 gcn4-101 his3-609 ura3-52 Dftr1::URA3 Dfre1::HIS3 Dfet4::TRP1, M3 MATalpha trp1-63 leu2-3,112 gcn4-101 his3-609 ura3-52 FRE1-HIS3::URA3 ctr1-3, FTRUNB1 MATa ino1-13 leu2-3,112 gcn4-101 his3-609 ura3-52 Dctr1::URA3. Yeast disruption mutant strains, YH001 and YH003, were made by homologous recombination. Yeast cells were grown in 1% yeast extract, 2% peptone, and 2% glucose (YPD), 1% yeast extract, 2% peptone, and 2% glycerol (YPG), and synthetic de ned medium (SD) supplied with appropriate amino acids. Two per cent agar was added for solid plate media (Sherman, 1991). For iron- or copper-depleted media, bathophenanthroline disulphonic acid disodium salt (BPDS) or bathocuproine disulphonic acid disodium salt (BPCS) (Wako Pure Chemical Industries, Ltd., Japan) was added, respectively. Functional expression in yeast A yeast expression vector (pyh23) was constructed as follows. The plasmid YEplac181 (Gietz and Sugino, 1988) was cleaved with XbaI and EcoRI, blunt ended with T4 DNA polymerase, and re-ligated in order to remove most of the multiple cloning sites. The HindIII site was similarly deleted. The plasmid was digested with SphI and the 728 bp fragment from pvt100u (Vernet et al., 1987) containing the ADH1 expression cassette was inserted. To insert a NotI site in the multicloning site of the cassette, the plasmid was cleaved with BamHI and blunt ended with T4 DNA polymerase and the phosphorylated linker AGCGGCCGCT (TaKaRa Shuzo, Japan) was inserted. The new plasmid pyh23 has HindIII, PvuII, PstI, XhoI, SstI, XbaI, and NotI sites in the ADH1 expression cassette. For constitutive expression of OsIRT1 in yeast cells, OsIRT1 cdna was inserted into the XhoI±NotI site in pyh23 to form pyh23-osirt1. Yeast transformation was carried out using the Li-acetate transformation method (Gietz and Schiestl, 1995). Results Cloning the cdna and genomic sequences of OsIRT1 The database contains expression sequence tag (EST) from rice (accession no. D49213) that is highly homologous to the metal transporter IRT1 from Arabidopsis (Eide et al., 1996). Speci c PCR primers were designed based on the EST sequence in order to isolate a corresponding cdna clone. These primers were used to amplify a fragment (280 bp) clone from cdna prepared from leaves of rice with 91% identity to the EST. The PCR-based strategy successfully allowed the isolation of a cdna clone identical to the ampli ed fragment from a cdna library made from Fe-de cient rice roots. This cdna clone had high sequence similarity to IRT1. The cdna was designated as OsIRT1. A rice genomic library was also screened by hybridization using the 280 bp ampli ed fragment as a probe. After several rounds of screening, a clone containing the genomic sequence of OsIRT1 cdna was isolated.

3 Cloning rice Fe(II) transporter 1679 Fig. 2. Growth of the Dftr1 Dfet4 Dfre1 yeast mutants on irondepleted media containing 50 mm BPDS, transformed with vector only (left) or pyh23-osirt1 (right). Fig. 1. Alignment of the deduced OsIRT1 protein and ZIP proteins from higher plants. Residues conserved in all sequences are shaded in gray. Slashes indicate a gap. Transmembrane domains and a variable region are indicated. The histidine-rich metal-binding domain is underlined. An arrowhead indicates the corresponding position of an intron in OsIRT1 gene. The rst (699 bp) and second (679 bp) exons of OsIRT1 cdna are separated by an intron (4.3 kb). Sequence of OsIRT1 cdna and the deduced amino acid sequence of OsIRT1 protein The OsIRT1 cdna is 1395 bp long and contains an open reading frame of 374 amino acids (accession no. AB070226). OsIRT1 protein has a strong similarity to the metal transport protein IRT1 from Arabidopsis (53% amino acid identity, accession no. U27590). IRT1 and related metal transporters comprise the ZIP metal transporter family (Guerinot, 2000). Within the ZIP family, Rit1, an iron transport protein from pea, has the highest amino acid identity to OsIRT1 (55%, AF065444). LeIRT1 and LeIRT2 from tomato have a strong similarity as well (55% and 55%, AF2(6266 and AF2(6266, respectively) (Eckhardt et al., 2001). The amino acid alignment of these proteins is shown in Fig. 1. The hydrophobicity pro le reveals that OsIRT1 has eight transmembrane domains with a `variable region' between domains 3 and 4. These are typical features found in the ZIP metal transporter family (Guerinot, 2000). The variable region was less similar among these proteins. The region of OsIRT1 contains a potential metal-binding domain rich in histidine residues, which was also found in IRT1. OsIRT1 has only one intron, while IRT1 has two. However, the position of the rst intron of IRT1 is same as OsIRT1 (Fig. 1). OsIRT1 reversed growth defect of ferrous uptake mutants of yeast The yeast strain YH003 (Dftr1 Dfet4 Dfre1) has disrupted null mutations in the genes for high- and low-af nity iron transporters and ferric reductase (Dancis et al., 1992; Dix et al., 1994; Stearman et al., 1996). YH003 could not be grown on SD media containing 50 mm of BPDS, a strong chelator of the ferrous iron. Heterologous expression of OsIRT1, however, reversed the growth defect of the mutant on iron-depleted media (Fig. 2). The effect of OsIRT1 expression in copper uptake mutant ctr1 was also checked. Two strains, M3 (ctr1-3) and FTRUNB1 (Dctr1) (Dancis et al., 1994) were tested. However, no signi cant effect on the growth defect of these strains could be found on media containing non-fermentable carbon source (YPG) or SD media containing 50 mm or 100 mm BPCS, a strong chelator of the copper ion (data not shown). Expression of OsIRT1 under trace metal nutrient de ciency Induction of OsIRT1 expression in rice roots and leaves under trace metal-limiting conditions was examined by Northern hybridization (Fig. 3). Transcripts of OsIRT1 from plants grown under nutrient-suf cient conditions were very faint in roots, and not visible in shoots. In roots, Fe-de ciency signi cantly increased the level of OsIRT1 transcripts. Cu-de ciency also increased the transcript level. Under Mn- and Zn-de ciency, induction was not observed. In shoots, however, no increase in transcript level was found under any metal de ciency conditions.

4 1680 Bughio et al. Fig. 3. Transcript levels of the OsIRT1 gene in the roots and leaves of rice plants grown under trace metal de ciency conditions. Ten mg of total RNA were used. `C' indicates plants grown in normal nutrient solution. Discussion In this paper, the isolation of a cdna and genomic clone of OsIRT1, an IRT1-like metal transport protein from rice is reported. OsIRT1 has features typical of the ZIP metal transporter family, such as eight transmembrane domains and a variable region with a histidine-rich metal binding domain (Guerinot, 2000). So far, several members of this family have been isolated from dicotyledonous species (Eckhardt et al., 2001; Eide et al., 1996; Vert et al., 2001). OsIRT1 is the rst member of the ZIP metal transporter family to be isolated from a graminaceous plant. It was also shown that OsIRT1 cdna reversed the growth defect of the yeast iron-uptake mutant ftr1 fet4 fre1 on iron-depleted media when expressed in the yeast cells. This suggests that OsIRT1 encodes a functional iron transporter. OsIRT1 transcripts were predominantly expressed in the roots, and were induced by Fe-de ciency. The fact that expression of OsIRT1 is limited to the roots and is induced by Fe-de ciency indicates the direct involvement of OsIRT1 in uptake of iron from soil. It has been reported that IRT1 is a metal transporter with a broad substrate speci city (Eide et al., 1996; Korshunova et al., 1999; Rogers et al., 2000). In addition to ferrous iron, IRT1 can transport manganese, zinc, and probably cadmium and cobalt, when expressed in yeast cells. LeIRT1 and LeIRT2 from tomato can also complement yeast mutants defective in the uptake of iron, zinc, manganese, and copper (Eckhardt et al., 2001). Although IRT1 has a broad substrate speci city, the transcript level was not increased by any of these metal de ciency conditions other than iron (Eide et al., 1996; Korshunova et al., 1999). Therefore, it is interesting that expression of OsIRT1 was also induced by Cu-de ciency in addition to Fe-de ciency. It was reported that LeIRT1 and LeIRT2 from tomato could reverse the growth defects of the yeast high-af nity copper uptake mutant, ctr1 (Dancis et al., 1994), while IRT1 from Arabidopsis could not (Eckhardt et al., 2001). Although no effects of OsIRT1 expression on the growth defects of ctr1 could be found in these experiments, OsIRT1 may participate in copper uptake when it is Cu-limited. The effects of OsIRT1 expression on manganese or zinc uptake yeast mutants were not tested. Although OsIRT1 was not induced by these metal de ciency conditions, it may have a broad substrate speci city for these metals similar to IRT1, LeIRT1, and LeIRT2. To date, it has been generally accepted that graminaceous plants can utilize only ferric iron in the rhizosphere in a phytosiderophore-dependent manner (RoÈmheld, 1987; RoÈmheld and Marschner, 1986). Compared with dicotyledonous plants, Fe-de ciency inducible Fe(II) transport systems are absent or expressed at lower levels in the roots of barley and maize (Zaharieva and RoÈmheld, 2001). This report on the isolation of a root-speci c IRT1-like Fe(II) transporter clearly demonstrates that rice possesses an iron uptake system for Fe(II), in addition to a phytosiderophore-mediated Fe(III) transport system. This is a new mechanism of iron uptake that combines both Strategy I and Strategy II. Rice is grown under submerged conditions in which the dominant form of soil iron is ferrous iron (Yoshida, 1981). Therefore, it seems reasonable that rice may have an iron uptake system for Fe(II) as well as Fe(III) in its roots. Rice also transports oxygen from the shoots to the roots, and secretes oxygen from roots to oxidize the rhizosphere. Therefore, rice plants may induce one or the other of these iron transport systems by sensing the concentration of ferrous iron or the redox potential around their rhizoplane. Perhaps rice is unique among graminaceous plants in possessing a transport system for Fe(II). There is another class of metal transporters, the Nramp family, involved in Fe(II) transport in higher plants. Nramp genes are widely distributed in mammals, yeasts, and higher plants, and are known to be involved in a variety of processes, including metal transport and resistance to microbial infection (Cellier et al., 1996; Cohen et al., 2000; Gunshin et al., 1997). AtNramp1 and OsNramp1 can complement the fet3 fet4 yeast mutant defective both in low- and high-af nity iron transporters. The expression of AtNramp1, however, did not enhance iron uptake in yeast mutant cells. These proteins were speculated to be localized to intracellular compartments, such as chloroplasts, and to facilitate intracellular iron transport (Curie et al., 2000). In another paper, it was reported that AtNramp3 and AtNramp4 could complement the yeast metal uptake-de cient strain smf1, in addition to the fet3 fet4 mutant. The authors suggested the involvement of Arabidopsis Nramp proteins in the transport of iron, manganese, and cadmium, including Fe-de ciency inducible iron transport, especially AtNramp3 (Thomine et al., 2000). There are at least three isologues of Nramp proteins in rice (Belouchi et al., 1997). However, the function of these proteins remains unclear. Recently, yellow stripe 1 (YS1), a strong candidate for the gene for the transport

5 protein for the ferric phytosiderophore complex, was isolated from maize (Curie et al., 2001). Several other rice YS1 homologues were found in the database. Further study might elucidate the function of each of these three classes of metal transport proteins (IRT, Nramp, and YS1), and how the expression of these proteins is regulated developmentally and spatially. Analysis of these genes will allow an understanding of iron nutrition from the viewpoint not only of iron uptake from soils, but iron transport in planta, as well as organellar iron transport. Acknowledgements The authors thank Dr Andrew Dancis for the gift of the yeast strains used for the complementation analysis. We also thank Dr Kyoko Higuchi for her gift of a cdna library and help interpreting data. This work was supported by the Japan Science and Technology Corporation (JST). 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