Iron (Fe) is essential for numerous biological oxidation–reduction reactions in plants. Despite its abundance in soils, Fe is present in the insoluble Fe(III) form under the oxidative soil environment and is not readily available to plants. The low solubility and availability of Fe in the soil solution often induces Fe deficiency in plants, especially in high-pH soils. Therefore, Fe deficiency is one of the biggest problems in crop production, reducing yields and quality. Higher plants have evolved two strategies to take up Fe from soils (Römheld and Marschner 1986). With the exception of graminaceous plants, higher plants reduce Fe3+ ions in soils using the ferric reductase FRO, and take up Fe2+ ions via the Fe2+ transporter IRT. Graminaceous plants absorb Fe3+ directly using the natural Fe3+ chelators named mugineic acid family phytosiderophores (MAs). MAs are biosynthesized in roots and secreted into the rhizosphere via a specific exporter of MAs (TOM1), and then the complex of Fe3+–MAs is taken up by an Fe3+–MAs transporter (YS1) on the surface of the root (Curie et al. 2001; Nozoye et al. 2011). TOM1, which encodes a major facilitator superfamily protein, was identified as a highly Fe deficiency-inducible gene in rice. Both TOM1 and its barley homolog, HvTOM1, cause the efflux of 2′-deoxymugineic acid (DMA), the primary molecule of the MAs (Nozoye et al. 2011). One of 18 members of YS1-like proteins in rice, OsYSL15, is involved in taking up Fe3+–DMA complexes from the rhizosphere, especially under Fe-deficient conditions (Inoue et al. 2009). The amount of MAs synthesized under Fe-sufficient conditions changes to meet plants’ growth requirements for Fe (Nomoto et al. 1987). Under Fe-deficient conditions, the amount of synthesized MAs increases dramatically. The synthesis of MAs is regulated at the transcriptional levels of the many types of genes that are related to the supply of the precursor of MAs and the actual synthesis of MAs (Higuchi et al. 19992001; Takahashi et al. 1999; Negishi et al. 2002; Inoue et al. 20032008; Kobayashi et al. 2005; Bashir et al. 2006). The methionine (Met) cycle, through which Met is supplied efficiently for ethylene production (Miyazaki and Yang 1987), also works for the production of MAs in graminaceous plants (Kobayashi et al. 2005; Suzuki et al. 2006). Promoter-GUS analyses revealed that in rice roots, the expression of MAs synthesis genes is increased by Fe deficiency in almost all tissues, whereas under Fe-sufficient conditions, their expression occurs mostly in vascular bundles (Inoue et al. 20032008; Bashir et al. 2006). Rice is also adapted to submerged paddy fields by taking up Fe2+ ions in the same manner as dicots and non-graminaceous monocots, in addition to the utilization of the Fe3+–MAs mechanism (Ishimaru et al. 2006). The expression of the rice Fe2+ transporter genes OsIRT1, OsIRT2, and OsNRAMP1 is induced under Fe-deficient conditions (Ishimaru et al. 2006; Takahashi et al. 2011a, b). In addition to MAs and their precursor nicotianamine (NA), citrate and phenolics, which can act as metal chelators, have been confirmed to be important for Fe homeostasis in rice (Yokosho et al. 2009; Ishimaru et al. 2011).
The transcriptional responses to Fe deficiency, including MAs synthesis and Fe uptake, are regulated by some specific cis elements, such as the Fe deficiency-responsive cis-acting elements (IDE) 1 and 2 (Kobayashi et al. 20032005). IDEF1 and IDEF2, which specifically bind to IDE1 and IDE2, respectively, were identified as key transcription activators in response to Fe deficiency in rice (Kobayashi et al. 20032007; Ogo et al. 2008). IDEF1 is a B3 region-containing protein, and IDEF2 is a NAC-domain protein. Both IDEF1 and IDEF2 themselves show no response to Fe deficiency at the transcriptional level (Kobayashi et al. 2007; Ogo et al. 2008). An Fe deficiency-inducible gene encoding a basic helix-loop-helix (bHLH) transcription factor, OsIRO2, is under the control of IDEF1 and regulates the expression of genes such as the NA synthase genes OsNAS1 and OsNAS2, OsYSL15, and TOM1 (Ogo et al. 200620072011; Kobayashi et al. 2007). Overexpression of OsIRO2 by the cauliflower mosaic virus 35S promoter results in increased OsIRO2 protein levels and high secretion of DMA from roots (Ogo et al. 2007). OsIRO2-overexpressing plants tolerated Fe deficiency in calcareous soils and grain yields were greater than for non-transformants, but no differences were observed between OsIRO2-overexpressing plants and non-transformants grown in normal soils (Ogo et al. 2011). Analysis of the OsIRO2-overexpressing plants indicated that transcriptional co-activators need to be activated by Fe deficiency to induce the expression of genes downstream of OsIRO2 (Ogo et al. 20072011). In contrast to OsIRO2, the regulation of MAs biosynthetic genes by IDEF1 is specifically restricted to early Fe deficiency than in the subsequent progressed state (Kobayashi et al. 2009). IDEF1 has characteristic histidine–asparagine repeat and proline-rich regions that are known for binding divalent metals including Fe2+. Through these metal-binding domains, the cellular metal ion balance caused by changes in Fe availability would be sensed by IDEF1 and switches the pattern of IDEF1 regulation from the early Fe-deficiency state to the progressed state (Kobayashi et al. 20092011).
The root is the organ where Fe uptake from the soils occurs, and it consumes Fe itself as well as sending Fe to the shoot. Most previous studies on Fe deficiency-induced genes have focused on the progressed Fe deficiency stage rather than on the onset of Fe deficiency. The primal responses to the onset of Fe deficiency at the transcriptional level are still unclear, not only in rice but also in other graminaceous plants. In this report, we carried out a 36-h time-course analysis of rice roots in the early stage of Fe deficiency using a 44 K microarray. We previously reported a time-course analysis of a 1-day span using a rice 22 K microarray, in which we discovered OsIRO2 (Ogo et al. 2006). The expression changes observed in the previous work were more dynamic in shoots than in roots. For analysis of the expression changes in roots, a much shorter time span seemed to be suitable. Therefore, a time-course analysis was performed in a 3-h span within 3–12 h after the onset of Fe-deficiency treatment, and in a 12-h span within 12–36 h after the onset of Fe-deficiency treatment. We report the synchronous expression of the MA biosynthetic genes, TOM1, OsYSL15, and OsIRO2, and the unique expression patterns of OsIDS1 and OsIRT1.