Iron is essential for most living organisms and is required for normal plant growth. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of this gene regulation system remain largely unknown. We identified the rice transcription factor IDEF1, which specifically binds the iron-deficiency-responsive cis-acting element IDE1. IDEF1 belongs to an uncharacterized branch of the plant-specific transcription factor family ABI3/VP1 and efficiently binds to the CATGC sequence within IDE1. IDEF1 transcripts are constitutively present in rice roots and leaves. Transgenic tobacco plants expressing IDEF1 under the control of the constitutive cauliflower mosaic virus 35S promoter transactivate IDE1-mediated expression only in iron-deficient roots. Transgenic rice plants expressing IDEF1 under the control of the iron-deficiency-inducible IDS2 promoter tolerate iron deficiency in hydroponic culture and calcareous soil. Conversely, transgenic rice plants with repressed IDEF1 expression are susceptible to early stage iron deficiency in hydroponic culture. Expression analysis of these transgenic plants revealed that IDEF1 positively regulates iron-deficiency-induced genes, including the ferrous iron transporter gene OsIRT1 and the iron-deficiency-induced transcription factor gene OsIRO2. These data suggest the presence of a sequential gene regulatory network that functions via novel cis element/trans factor interactions to promote the iron-deficiency response.

A highly sensitive quantitative method for assaying nicotianamine (NA) and 2'-deoxymugineic acid (DMA) using liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) was developed. This method requires fewer sample preparation steps than high-performance liquid chromatography (HPLC). The amino and hydroxyl groups of NA and DMA were derivatized using 9-fluorenylmethoxycarbonyl chloride, which improved their retention on a C18 reversed-phase column and facilitated separation. The amounts of NA and DMA in 10 μl of xylem sap from rice cultivated under Fe-sufficient and Fe-deficient conditions were quantified without concentration. In Fe-sufficient plants, the concentrations of NA and DMA were almost equal to that of Fe. In Fe-deficient plants, the concentration of NA did not change significantly, whereas that of DMA increased markedly.

Cadmium (Cd) is one of the most toxic heavy metals for both plants and humans, and Cd accumulation in rice grains has become a major agricultural problem in Japan. In this study, we investigated the role of OsNramp1 in Cd uptake and transport in rice. An OsNramp1::GFP fusion protein localized to the plasma membrane in onion epidermal cells, and yeast expressing OsNramp1 showed increased sensitivity to Cd. Furthermore, OsNramp1-overexpressing rice accumulated more Cd in the leaf blades than non-transformed rice when cultured in Cd-contaminated soil. These results suggest that OsNramp1 increases the cellular uptake and accumulation of Cd in plants.

Iron (Fe) is critical for plant growth. Under conditions of low Fe availability, rice plants induce genes involved in Fe uptake and utilization. Recently, we identified the iron-deficiency-responsive cis-acting elements IDE1 and IDE2, which are bound in a specific manner by IDE-binding factors 1 and 2 (IDEF1 and IDEF2, respectively). IDEF1 and IDEF2 regulate genes related to the Fe-deficiency response. In the present study, we examined the spatial expression patterns of IDEF1 and IDEF2 during the germination, vegetative, and seed-maturation stages by assessing their localization in IDEF1 and IDEF2 promoter–GUS transgenic rice lines. During germination, IDEF1 and IDEF2 were expressed in the endosperm and embryo. In hydroponic culture experiments, the expression patterns of IDEF1 and IDEF2 were similar under both Fe-deficient and Fe-sufficient conditions during the vegetative stage. In leaves, IDEF1 expression was observed in mesophyll cells and in small vascular bundles, whereas IDEF2 was highly expressed in vascular bundles. In root sections, IDEF1 and IDEF2 were highly expressed in secondary roots and inside vascular bundles. IDEF1 was also expressed in pollen from flowers before and after anthesis, in the ovary after fertilization, and in the embryo during seed maturation. IDEF2 was expressed in flowering stage pollen, in immature seeds just after flowering, and in the dorsal vascular region during the late maturation stage. The spatial expression patterns of IDEF1 and IDEF2 partially overlapped with their target genes.

Iron deficiency is a worldwide agricultural problem on calcareous soils with low iron (Fe) availability and also poses a major human nutritional problem. Plants induce Fe-acquisition systems under conditions of low Fe availability. Previously, we reported that an Fe deficiency-inducible bHLH transcription factor, OsIRO2, is responsible for regulating the genes involved in Fe homeostasis in rice. Here, we show that OsIRO2 overexpression results in improved tolerance to low Fe availability in calcareous soil. In addition to increased Fe content in shoots, rice overexpressing OsIRO2 accumulates more Fe in seeds than non-transformant rice when grown on calcareous soil. OsIRO2 promoter–GUS analysis revealed that OsIRO2 expression could be detected in developing seeds. These results suggest that OsIRO2 plays a crucial role in regulating both Fe uptake from soil and Fe translocation to grain. Improved uptake and translocation of Fe in the OsIRO2-overexpressing rice under low Fe availability provides an approach for producing graminaceous crops that are tolerant to Fe deficiency and have Fe-rich seeds to enhance production and the nutritional quality of food.

Mitochondrial iron (Fe) homeostasis is important for various cellular processes including heme synthesis and Fe-S cluster synthesis, however the proteins involved in mitochondrial Fe uptake and homeostasis has not been characterized in plants. We have cloned and characterized a putative Mitochondrial Iron Transporter in rice (OsMIT). OsMIT was identified by T-DNA knockout mutant screening as a member of mitochondrial substrate carrier family and is homologous to yeast mitochondrial Fe transporters MRS3 and MRS4. OsMIT is located on rice chromosome 3 and encodes a predicted polypeptide of 329 amino acids. OsMIT-GFP protein, when expressed in tobacco BY-2 cells, was localized to the mitochondria. Yeast mrs3mrs4 double knockout mutants were used to investigate the role of OsMIT. Overexpression of OsMIT in yeast complemented the growth defect of yeast strain mrs3mrs4 under conditions of limiting Fe confirming that OsMIT plays a role in mitochondrial Fe homeostasis. Moreover, the mrs3mrs4 yeast strain expressing OsMIT accumulated comparable levels of Fe to WT in contrast to vector control which accumulated high Fe compared to WT. These results suggested that OsMIT is a mitochondrial protein involved in Fe homeostasis. The characterization of OsMIT will provide the chances to increase our understanding about mitochondrial Fe homeostasis and enable us to develop strategies to mitigate Fe deficiency stress in plants.

Iron (Fe) is essential for most living organisms, and thus Fe deficiency poses a major abiotic stress in crop production. Plants induce Fe utilization systems under conditions of low-Fe availability, but the molecular mechanisms of gene regulation under Fe deficiency remain largely unknown. We identified a novel transcription factor in rice and barley, IDEF2, which specifically binds to the Fe deficiency-responsive cis-acting element 2 (IDE2), using yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets (CASTing) experiment revealed that IDEF2 predominantly recognizes CA[A/C]G[T/C][T/C/A][T/C/A] within IDE2 as the core binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by RNA interference (RNAi) and chimeric repressor gene-silencing technology (CRES-T) caused aberrant Fe distribution between shoots and roots in rice grown hydroponically under Fe-sufficient or deficient conditions. These results reveal novel cis-element/trans-factor interactions that are functionally associated with iron homeostasis.

Mitochondria utilize iron (Fe), but the proteins involved in mitochondrial Fe regulation have not been characterized in plants. We cloned and characterized a mitochondrial iron-regulated (MIR) gene that is involved in Fe homeostasis in rice. MIR expressed in tobacco BY-2 cells was localized to the mitochondria. MIR transcription was significantly increased in response to Fe deficiency in the roots and shoots. MIR is not homologous to any known protein, as no homologs were found in the rice or Arabidopsis genome databases, or in the EST database for other organisms. Growth in the MIR T-DNA knock-out rice mutant (mir) was significantly impaired compared with wild-type (WT) plants, under both Fe-sufficient and Fe-deficient conditions. Furthermore, Fe accumulation in shoots and roots of mir plants was more than twice that in WT plants, under both Fe-sufficient and Fe-deficient conditions. Despite the high accumulation of Fe in roots and shoots, Fe deficiency-inducible genes were expressed in mir plants, indicating that mir may not be able to utilize the Fe for physiological functions. These results suggest that MIR is a rice-specific mitochondrial protein that plays a significant role in Fe homeostasis.