Molecular Plant Advance Access originally published online on July 22, 2009
Molecular Plant 2009 2(5):1059-1066; doi:10.1093/mp/ssp051
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Rice-Specific Mitochondrial Iron-Regulated Gene (MIR) Plays an Important Role in Iron Homeostasis
a Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan
b Department of Life Sciences, Pohang University of Science and Technology, Pohang, 790–784, Republic of Korea
c Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Ishikawa 921-8836, Japan
1 To whom correspondence should be addressed. E-mail annaoko{at}mail.ecc.u-tokyo.ac.jp, fax +81 3 5841 7514, tel. +81 3 5841 7514.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTS AND DISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Mitochondria utilize iron (Fe), but the proteins involved in mitochondrial Fe regulation are not characterized in plants. We cloned and characterized a mitochondrial iron-regulated (MIR) gene in rice involved in Fe homeostasis. MIR, when expressed in tobacco BY-2 cells, was localized to the mitochondria. MIR transcripts were greatly increased in response to Fe deficiency in roots and shoot tissue. MIR is not homologous to any known protein, as homologs were not found in the rice or Arabidopsis genome databases, or in the EST database for other organisms. Growth in the MIR T-DNA knockout rice mutant (mir) was significantly impaired compared to wild-type (WT) plants when grown under Fe-deficient or -sufficient conditions. Furthermore, mir plants accumulated more than twice the amount of Fe in shoot and root tissue compared to WT plants when grown under either Fe-sufficient or -deficient conditions. Despite the high accumulation of Fe in roots and shoots, mir plants triggered the expression of Fe-deficiency-inducible genes, indicating that mir may not be able to utilize Fe for physiological functions. These results clearly suggest that MIR is a rice-specific mitochondrial protein, recently evolved, and plays a significant role in Fe homeostasis.
Key Words: Fe-deficiency-regulated gene • Fe homeostasis • mitochondria • Oryza sativa
Received for publication April 29, 2009. Accepted for publication June 23, 2009.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Iron (Fe) is an essential micronutrient for plants and plant growth, and development is significantly impaired under Fe-deficient conditions. In plants, Fe is essential for several cellular processes, such as respiration, chlorophyll biosynthesis, and photosynthetic electron transport (Marschner, 1995). In mitochondria, Fe is essential for the synthesis of heme by ferrochelatase in the matrix, and for the synthesis of several Fe–sulfur cluster-containing proteins (Fe–S proteins) of both the matrix (e.g. aconitase and homoaconitate hydratase) and the inner membrane (Rieske Fe–S protein). Limiting Fe content impairs the metabolic and respiratory activities of the organelle, whereas excess Fe may exert toxic effects by the generation of oxidative stress through the formation of radicals. Free Fe ions might be particularly harmful to mitochondria, where free reactive oxygen species are generated as a side reaction of electron transport. In yeast, the alteration of mitochondrial activity by the inhibition of mitochondrial Fe transport leads to Fe accumulation in vacuoles and Fe deficiency in the cytoplasm (Li and Kaplan, 2004). In plants, some of the genes related to heme synthesis, Fe–sulfur cluster synthesis, and the Fe–S cluster efflux transporter have been characterized, but others are still unknown.
Rice plants use two pathways to absorb Fe from the soil. First, Fe (II) is absorbed from the soil by Fe2+ transporters (Ishimaru et al., 2006). Second, rice plants synthesize and secrete 2'-deoxymugineic acid (DMA) to chelate Fe3+. DMA is synthesized from methionine (Mori and Nishizawa, 1987), which is converted into S-adenosyl-L-methionine (SAM) by SAM synthase (SAMS). Subsequently, three molecules of SAM are combined to form one molecule of nicotianamine (NA) by NA synthase (NAS). NA is then converted to a 3"-keto acid by NA aminotransferase (NAAT), and DMA is synthesized through DMA synthase (DMAS). The expression of OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1 is induced in both roots and shoots under Fe-deficient conditions (Higuchi et al., 2001; Inoue et al., 2003, 2008; Bashir et al., 2006). In addition, the transporters involved in the absorption and translocation of Fe in rice have been characterized. OsYSL15 (Inoue et al., 2009) and OsIRT1 (Bughio et al., 2002) were reported to be Fe (III)-MAs and Fe (II) transporters, respectively, which take up Fe from the rhizosphere. OsYSL2 is an Fe (II)-NA and manganese (Mn) (II)-NA transporter responsible for the phloem transport of Fe and Mn (Koike et al., 2004). The expression of these biosynthetic genes and transporters is greatly increased in response to Fe deficiency.
Recently, the Fe-deficiency-responsive cis-acting elements IDE1 and IDE2 were identified (Kobayashi et al., 2003), and IDE-binding factors IDEF1 and IDEF2 were characterized in rice (Kobayashi et al., 2007; Ogo et al., 2008). IDEF1 recognizes the CATGC sequence within IDE1, whereas IDEF2 predominantly recognizes CA[A/C]G[T/C][T/C/A][T/C/A] within IDE2 as the core binding site. IDEF2 regulates OsYSL2, and IDEF1 regulates OsIRT1 and the Fe-deficiency-induced bHLH transcription factor gene, OsIRO2 (Ogo et al., 2007). Despite rapid progress in understanding Fe-deficiency responses in rice, the factors involved in mitochondrial Fe homeostasis have not been characterized.
In this study, we cloned and characterized a mitochondrial iron-regulated gene in rice (MIR), which was highly up-regulated in Fe-deficient roots and shoots. Interestingly, there were no homologs of MIR in the rice and Arabidopsis genome databases or in the EST database for other organisms. MIR was localized to the mitochondria, and its transcripts were greatly increased by Fe deficiency. The MIR T-DNA knockout rice mutant (mir) constitutively induced the genes involved in Fe-deficiency response, as if the plants were consistently Fe-deficient in spite of higher Fe accumulation compared to wild-type (WT) plants. These data suggest that MIR is a rice-specific gene and plays a significant role in rice Fe homeostasis via the mitochondrion.
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RESULTS AND DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Cloning of MIR
MIR (Os12g0282000; AK103636 [GenBank] ) was identified through an oligo microarray analysis, as its expression was highly up-regulated in response to Fe deficiency in roots as well as in shoots (data not shown). MIR is located on rice chromosome 12, is composed of three exons, and contains 10 copies of the IDE1 core sequence (CATGC) within 1 kb of the promoter region (Figure 1A). As IDE1 plays an important role in triggering plant Fe-deficiency response (Kobayashi et al., 2003, 2007), the presence of a high number of IDE1 binding sequences in the MIR promoter region shows that the expression of MIR is regulated by Fe availability, and, at the same time, indicates its importance in Fe homeostasis in rice.
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Characterization of the MIR Knockout Mutant (MIR)
To characterize the role of MIR, the T-DNA knockout mutant of MIR was obtained from the rice functional genomics database maintained at http://signal.salk.edu/cgi-bin/RiceGE. According to the database, the T-DNA is integrated into the third exon (Figure 1A). The integration of T-DNA was confirmed by polymerase chain reaction (PCR) using internal primers for T-DNA and MIR (Figure 1B). When MIR-specific primers were used, a clear band was observed in WT plants whereas no band was observed in mir plants due to the integration of the T-DNA. RT–PCR further confirmed that MIR was not expressed in mir plants grown under Fe-sufficient or -deficient conditions (Figure 1C).
Quantitative real time reverse transcriptase (RT)–PCR analysis revealed that transcripts of MIR in Fe-deficient WT plants were more abundant compared to those in Fe-sufficient WT roots and shoots (Figure 2A). These results agree with the microarray analysis, confirming that MIR is highly up-regulated by Fe-deficiency stress in roots and shoots (Table 1). The growth of mir plants was significantly impaired compared to WT plants, when plants were grown hydroponically in the presence or absence of Fe (Figure 2B). The shoot length was 13.8 and 18.2% less (Figure 2C), and root length was reduced by 24 and 43% compared to WT plants, when plants were cultured in the presence or absence of Fe, respectively (Figure 2D). The shoot dry weight was 57% less compared to WT plants, regardless of whether the plants were grown in the presence or absence of Fe (Figure 2E). The root dry weight was also significantly reduced, by 73 and 67%, when plants were cultured in the presence or absence of Fe, respectively (Figure 2F). Although mir plants had an abnormal phenotype in the presence of Fe, the abnormalities were more severe with respect to root and shoot length when plants were grown without Fe. When grown in soil, mir plants were mostly sterile and set only a few seeds (data not shown).
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Compared to WT plants grown under the same conditions, mir plants accumulated high Fe concentrations in root and shoots tissue. In the shoots of mir plants, Fe concentration was 2.3 and 2.2 times higher compared to WT plants grown under Fe-sufficient and Fe-deficient conditions, respectively (Figure 3A). Furthermore, mir plants grown under Fe-deficient conditions accumulated as much Fe as did the WT plants grown under Fe-sufficient conditions, yet still showed symptoms of Fe deficiency (Figure 2C). These results indicate that although mir plants had enough Fe in shoot tissues, this Fe was probably not available for physiological functions, resulting in symptoms typical of Fe-deficiency stress. The concentrations of other metals like Zn, Mn, or Cu were not significantly affected in mir plants compared to WT plants (Figure 3B–3D). Root Fe concentration was also significantly higher compared to WT plants grown under the same conditions. When grown under Fe-sufficient and Fe-deficient conditions, mir plants accumulated 2.2 and 2.8 times more Fe compared to WT plants, respectively (Figure 3E). The concentration of Zn and Mn in mir roots was also higher than in WT roots (Figure 3F and 3G), whereas the concentration of Cu was comparable to that in WT roots (Figure 3H). Soil-grown mir plants accumulated more Fe and Cu in seeds compared to WT seeds (Figure 3I).
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The 44K microarray was used to examine changes in gene expression in WT (Wt –/+) and mir (–/+) plants grown under Fe-sufficient and -deficient conditions (Table 1). These results confirmed that, in WT plants, MIR is significantly up-regulated under Fe-deficient conditions. These results also confirmed the knockout status of the mir plants. Furthermore, microarray analysis revealed that, in mir roots grown under Fe-sufficient conditions, the expression of genes normally up-regulated in response to Fe deficiency, such as OsYSL2, OsYSL15, OsIRT1, OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1, were significantly increased compared to WT plants (mir/WT, +root) (Table 1). OsNAAT1, OsFRO2, and OsNramp1 were up-regulated in mir shoots (mir/WT, +shoot). OsNramp1 is an Fe (II) transporter (Curie et al., 2000), whereas OsFRO2 is a homolog of Arabidopsis ferric chelate reductase (FRO), responsible for the reduction of Fe (Robinson et al., 1999). When plants were grown without Fe, only the expressions of OsNAS1, OsNAS2, and OsDMAS1 were higher in mir roots compared to WT roots, whereas the expression profile of mir shoots grown without Fe was comparable to that of WT plants grown under similar conditions (Table 1). The complete list of genes up-regulated in response to Fe deficiency in WT and mir plants can be seen in Supplemental Table 1.
The up-regulation of these genes appears to be responsible for the high accumulation of Fe in mir plants, and, at the same time, indicates that this Fe may not be available for physiological functions, as plant Fe-deficiency responsive genes are only up-regulated under Fe-deficient conditions (Higuchi et al., 2001; Inoue et al., 2003, 2008; Bashir et al., 2006). Plants primarily store Fe in ferritin, a multimeric protein that can store up to 4500 atoms of Fe, which plays an important role in Fe homeostasis (Harrison and Arosio, 1996). The expression of ferritin increases with Fe availability to avoid cellular damage and decreases when Fe is deficient (Briat et al., 1999). Microarray analysis did not reveal any difference in the expression of ferritin in mir or WT plants, raising the possibility that the Fe in mir plants is not stored in ferritin, as Fe stored in ferritin is bio-available under conditions of Fe deficiency. Thus, the possible reason for this unavailability may be that Fe is present in a form that cannot be easily used by plants.
Sub-Cellular Localization of MIR
In silico analysis using PSORT (http://psort.ims.u-tokyo.ac.jp/) indicated the possibility that it may be a cytoplasmic protein. However, MIR-sGFP, when transiently expressed in tobacco BY-2 cells, co-localized with cells stained with MitoTracker (Figure 4A–4C). Some proteins can co-localize to mitochondria and chloroplast, such as AtSufE, a protein involved in the synthesis of Fe–sulfur cluster, localizes to mitochondria and chloroplast (Xu and Møller, 2006). As the tobacco BY-2 cells do not have chloroplast, MIR was also transiently expressed in tulip (Tulipa gesneriana) guard cells to check if the MIR co-localizes to chloroplast. In tulip, it localized as a small spot distinct from chloroplast auto-fluorescence, confirming that MIR is localized to mitochondria, and not to chloroplast (Figure 5A–5C).
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MIR, a Rice-Specific Gene Important to Fe Homeostasis
In silico analyses were performed to identify MIR paralogs and orthologs. Although there is a conserved sequence in rice, similar to MIR (Os04g0192200; J065068L22), it is a non-protein coding transcript; therefore, MIR exists as a single-copy gene in the rice genome. MIR orthologs were not found in the Arabidopsis genome database or the EST database for other organisms (TAIR, www.arabidopsis.org/; National Center for Biotechnology Information, www.ncbi.nlm.nih.gov/; TIGR plant transcript assemblies, http://plantta.jcvi.org/). In silico analysis indicated that it may be a soluble protein and it does not contain any preserved domain or conserved sequence found in any known genome. Moreover, the expression of MIR orthologs in barley was not detected through Northern blot analysis in Fe-sufficient or -deficient root and shoot samples when MIR was used as a probe (data not shown). These results indicate that MIR is a recently evolved, rice-specific gene. As MIR is not homologous to any known protein and does not contain any conserved domains, it is difficult to predict its function. However, its importance in Fe-deficiency response cannot be overlooked, as MIR is clearly induced by Fe-deficiency stress and Fe homeostasis is significantly impaired in the mir knockout mutant. As MIR is localized to the mitochondrion, it appears to play a role in mitochondrial Fe homeostasis. Microarray analysis did not indicate any change in the expression of genes involved in Fe–S cluster synthesis or other proteins in mitochondrial Fe homeostasis. Mitochondrial Fe regulation is poorly understood in rice, and many factors have yet to be identified. Thus, the characterization of MIR provides an opportunity to examine Fe homeostasis in rice and to develop strategies to mitigate this problem.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Cloning of MIR
Full-length rice cDNA clone for MIR (Os12g0282000; AK103636 [GenBank] ) was acquired from the rice full-length cDNA database (KOME; http://cdna01.dna.affrc.go.jp/cDNA/). The information about genomic structure of this clone was collected from the RAP-DB rice genome database (http://rapdb.dna.affrc.go.jp/). To find MIR orthologs or paralogs, the web-based applications maintained at TAIR, www.arabidopsis.org/; National Center for Biotechnology Information, www.ncbi.nlm.nih.gov/; TIGR plant transcript assemblies, http://plantta.jcvi.org/, were utilized.
RT–PCR
Total RNA was isolated from mir and WT plants grown under Fe-sufficient or -deficient conditions, First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen, Japan) by priming with oligo-d(T)30. cDNA was amplified by PCR in a Smart Cycler (Takara, Japan) with SYBR Green I and ExTaqTM RT–PCR Version X (Takara, Japan). The primers used for RT–PCR were MIR forward 5'- CGTCATGGTCTTCGGTCTCCTACGTGCTCG-3’ and MIR reverse 5'- GCTAGTCGTTGTCAACAGTCAACACAAAGA-3'. The 
-tubulin primers used for RT–PCR were -tubulin forward 5'-TCTTCCACCCTGAGCAGCTC-3’ and -tubulin reverse 5'-AACCTTGGAGACCAGTGCAG-3'.
Oligo-DNA Microarray Analysis
The MIR T-DNA knockout line (mir) was obtained from the rice functional genomics database maintained at http://signal.salk.edu/cgi-bin/RiceGE. Isolation of mutant plant was performed by PCR-based screening using T-DNA right border specific primers 5'-GTTACGTCCTGTAGAAACCCCAACCC-3’ and 5'-ATACGCTGGCCTGCCCAACCTTTCG-3'. Moreover, MIR internal primers 5'-CGTCATGGTCTTCGGTCTCCTACGTGCTCG-3’ and 5'-GCTAGTCGTTGTCAACAGTCAACACAAAGA-3', located outside the T-DNA integration site, were used to confirm the T-DNA integration site and homozygous status of mir. Rice seeds were germinated on wet filter paper and cultured as described (Inoue et al., 2003). For Fe-deficiency treatments, plants were transferred to culture solution lacking Fe. Roots and leaves were harvested after 7 d, frozen in liquid nitrogen, and stored at –80°C until use. RNA was extracted from the roots and shoots of three plants and total RNA samples (200 ng) from mir and WT plants were labeled with Cy3 or Cy5 using the Agilent Low RNA Input Fluorescent Linear Amplification Kit and microarray analysis were performed in duplicate according to the manufacturer's instructions using rice 44K oligo-DNA microarray (Agilent Technologies, CA). The point showing the signal value of Fe-deficiency-treated plants >500, the P-value <0.001, and the ratio >2 in both Cy3 and Cy5 channels was considered as significantly up-regulated.
Determination of Metal Concentrations
The elemental analysis of the WT and mir knockout plants was performed using inductively coupled plasma atomic emission spectrometry (SPS1200VR; Seiko, Japan) as described previously (Ishimaru et al., 2007).
Sub-Cellular Localization of MIR
The full-length ORF of MIR was amplified with forward and reverse primers as 5'-caccctcgagATGCCCGTGACTCAGCACTTAGGCA-3’ and 5'-ccccccgtcgacACAGTCAACACAAAGAGAAGCAC-3’ containing Xho1 and Sal1 restriction sites, respectively, and was subcloned into CaMV35S-SalI-KpnI-sGFP(S65T)-NOS3 as described (Ishimaru et al., 2006). Tulip leaf epidermal cells were transformed and observed with a LSM5 Pascal laser-scanning confocal microscope (Carl Zeiss, Japan) as described previously (Ishimaru et al., 2006).
Further, full-length MIR was cloned in to pENTR/D-TOPO (Invitrogen) and then subcloned into pH7WGF2 (Karimi et al., 2002) using LR recombination reaction (Invitrogen, Japan). BY-2 cells were bombarded using a PDS-1000 particle delivery system (Bio-Rad, Richmond, CA) as described previously Nakazono et al., 2000) and examined as described (Li et al., 2000).
Statistical Analysis
To determine whether the observed difference between WT and mir plants were statistically significant, one-way ANOVA was performed with completely randomized design followed by Student–Newman–Keuls test (p < 0.05).
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SUPPLEMENTARY DATA |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTS AND DISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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No conflict of interest declared.
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Acknowledgements |
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We are thankful to Dr. Yoshiaki Nagamura (the Rice Genome Project and the NIAS DNA Bank) for microarray analysis. We are also thankful to our colleagues for useful discussions and continuous support. This research was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to N.K Nishizawa.
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Notes |
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2 These authors contributed equally to this work.
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