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.

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.