Reiko Nakanishi Itai

A Novel NAC Transcription Factor, IDEF2, That Recognizes the Iron Deficiency-responsive Element 2 Regulates the Genes Involved in Iron Homeostasis in Plants

Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by 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 experiment revealed that IDEF2 predominantly recognized 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 the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.

The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants

Iron is essential for most living organisms and is often the major limiting nutrient for normal growth. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency 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 exhibits the sequence recognition property of efficiently binding 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 an introduced IDEF1 exhibit substantial tolerance to iron deficiency in both hydroponic culture and calcareous soil. IDEF1 overexpression leads to the enhanced expression of the iron deficiency-induced transcription factor gene OsIRO2, suggesting the presence of a sequential gene regulatory network. These findings reveal cis element/trans factor interactions that are functionally linked to the iron deficiency response. Manipulation of IDEF1 also provides another approach for producing crops tolerant of iron deficiency to enhance food and biomass production in calcareous soils.

The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes

Higher plants maintain iron homeostasis by regulating the expression of iron (Fe)-related genes in accordance with Fe availability. The transcription factor IDEF1 regulates the response to Fe deficiency in Oryza sativa (rice) by recognizing CATGC sequences within the Fe deficiency-responsive cis-acting element IDE1. To investigate the function of IDEF1 in detail, we analyzed the response to Fe deficiency in transgenic rice plants exhibiting induced or repressed IDEF1 expression. Fe-deficiency treatment in hydroponic culture revealed that IDEF1 knock-down plants are susceptible to early-stage Fe deficiency, in contrast to IDEF1-induced plants. Time-course expression analyses using quantitative reverse-transcriptase PCR revealed that the IDEF1 expression level was positively correlated with the level of induction of the Fe utilization-related genes OsIRO2, OsYSL15, OsIRT1, OsYSL2, OsNAS1, OsNAS2, OsNAS3 and OsDMAS1, just after the onset of Fe starvation. However, this overall transactivation mediated by IDEF1 became less evident in subsequent stages. Microarray and in-silico analyses revealed that genes positively regulated by IDEF1, especially at the early stage, exhibit over-representation of CATGC and IDE1-like elements within the proximal promoter regions. These results indicate the existence of early and subsequent responses to Fe deficiency, with the former requiring IDEF1 more specifically. Proximal regions of IDEF1-regulated gene promoters also showed enrichment of RY elements (CATGCA), which regulate gene expression during seed maturation. The expression of several genes encoding late embryogenesis abundant proteins, including Osem, was induced in Fe-deficient roots and/or leaves in an IDEF1-dependent manner, suggesting a possible function of seed maturation-related genes in Fe-deficient vegetative organs.

OsIRO2 is responsible for iron utilization in rice and improves growth and yield in calcareous soil

Iron (Fe) deficiency, a worldwide agricultural problem on calcareous soil with low Fe availability, is also a major human nutritional deficit. Plants induce Fe acquisition systems under conditions of low Fe availability. Previously, we reported that an Fe-deficiency-inducible basic helix-loop-helix (bHLH) transcription factor, OsIRO2, is responsible for regulation of the genes involved in Fe homeostasis in rice. Using promoter-GUS transformants, we showed that OsIRO2 is expressed throughout a plant’s lifetime in a spatially and temporally similar manner to the genes OsNAS1, OsNAS2 and TOM1, which is involved in Fe absorption and translocation. During germination, OsIRO2 expression was detected in embryos. OsIRO2 expression in vegetative tissues was restricted almost exclusively to vascular bundles of roots and leaves, and to the root exodermis under Fe-sufficient conditions, and expanded to all tissues of roots and leaves in response to Fe deficiency. OsIRO2 expression was also detected in flowers and developing seeds. Plants overexpressing OsIRO2 grew better, and OsIRO2-repressed plants showed poor growth compared to non-transformant rice after germination. OsIRO2 overexpression also resulted in improved tolerance to low Fe availability in calcareous soil. In addition to increased Fe content in shoots, the overexpression plants accumulated higher amounts of Fe in seeds than non-transformants when grown on calcareous soil. These results suggest that OsIRO2 is synchronously expressed with genes involved in Fe homeostasis, and performs a crucial function in regulation not only of Fe uptake from soil but also Fe transport during germination and Fe translocation to grain during seed maturation.

Iron-binding haemerythrin RING ubiquitin ligases regulate plant iron responses and accumulation

Iron is essential for most living organisms. Plants transcriptionally induce genes involved in iron acquisition under conditions of low iron availability, but the nature of the deficiency signal and its sensors are unknown. Here we report the identification of new iron regulators in rice, designated Oryza sativa Haemerythrin motif-containing Really Interesting New Gene (RING)- and Zinc-finger protein 1 (OsHRZ1) and OsHRZ2. OsHRZ1, OsHRZ2 and their Arabidopsis homologue BRUTUS bind iron and zinc, and possess ubiquitination activity. OsHRZ1 and OsHRZ2 are susceptible to degradation in roots irrespective of iron conditions. OsHRZ-knockdown plants exhibit substantial tolerance to iron deficiency, and accumulate more iron in their shoots and grains irrespective of soil iron conditions. The expression of iron deficiency-inducible genes involved in iron utilization is enhanced in OsHRZ-knockdown plants, mostly under iron-sufficient conditions. These results suggest that OsHRZ1 and OsHRZ2 are iron-binding sensors that negatively regulate iron acquisition under conditions of iron sufficiency.

The rice transcription factor IDEF1 directly binds to iron and other divalent metals for sensing cellular iron status

Iron is essential for most living organisms and its availability often determines survival and proliferation. The Oryza sativa (rice) transcription factor IDEF1 plays a crucial role in regulating iron deficiency-induced genes involved in iron homeostasis. In the present report, we found characteristic histidine-asparagine repeat and proline-rich regions in IDEF1 and its homolog in Hordeum vulgare (barley), HvIDEF1. An immobilized metal ion affinity chromatography assay revealed that IDEF1 and HvIDEF1 bind to various divalent metals, including Fe(2+) and Ni(2+) . Recombinant IDEF1 protein expressed in Escherichia coli contained mainly Fe and Zn. This metal-binding activity of IDEF1 was almost abolished by deletion of the histidine-asparagine and proline-rich regions, but DNA-binding and trans-activation functions were not impaired by the deletion. Transgenic rice plants constitutively overexpressing IDEF1 without these metal-binding domains failed to cause pleiotropic effects conferred by overexpression of full-length IDEF1, including a low germination rate, impaired seedling growth, tolerance to iron deficiency in hydroponic culture, and enhanced expression of various iron deficiency-inducible genes. Impairment of the transcriptional regulation of IDEF1 by deletion of the metal-binding domains occurred primarily at an early stage of iron deficiency. These results suggest that the histidine-asparagine and proline-rich regions in rice IDEF1 directly bind to divalent metals and sense the cellular metal ion balance caused by changes in iron availability.

Expression and enzyme activity of glutathione reductase is upregulated by Fe-deficiency in graminaceous plants.

Glutathione reductase (GR) plays an important role in the response to biotic and abiotic stresses in plants. We studied the expression patterns and enzyme activities of GR in graminaceous plants under Fe-sufficient and Fe-deficient conditions by isolating cDNA clones for chloroplastic GR (HvGR1) and cytosolic GR (HvGR2) from barley. We found that the sequences of GR1 and GR2 were highly conserved in graminaceous plants. Based on their nucleotide sequences, HvGR1 and HvGR2 were predicted to encode polypeptides of 550 and 497 amino acids, respectively. Both proteins showed in vitro GR activity, and the specific activity for HvGR1 was 3-fold that of HvGR2. Northern blot analyses were performed to examine the expression patterns of GR1 and GR2 in rice (Os), wheat (Ta), barley (Hv), and maize (Zm). HvGR1, HvGR2, and TaGR2 were upregulated in response to Fe-deficiency. Moreover, HvGR1 and TaGR1 were mainly expressed in shoot tissues, whereas HvGR2 and TaGR2 were primarily observed in root tissues. The GR activity increased in roots of barley, wheat, and maize and shoot tissues of rice, barley, and maize in response to Fe-deficiency. Furthermore, it appeared that GR was not post-transcriptionally regulated, at least in rice, wheat, and barley. These results suggest that GR may play a role in the Fe-deficiency response in graminaceous plants.

Rice-Specific Mitochondrial Iron-Regulated Gene (MIR) Plays an Important Role in Iron Homeostasis

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.

Iron deficiency responses in rice roots

Iron (Fe) is an essential element for most living organisms. To acquire sparingly soluble Fe from the rhizosphere, rice roots rely on two Fe acquisition pathways. The first of these pathways involves Fe(III) chelators specific to graminaceous plants, the mugineic acid family phytosiderophores, and the second involves absorption of Fe2+. Key components in this response include enzymes involved in the biosynthesis of deoxymugineic acid (OsNAS1, OsNAS2, OsNAAT1, and OsDMAS1), the deoxymugineic acid efflux transporter (TOM1), the Fe(III)-deoxymugineic acid transporter (OsYSL15), and Fe2+ transporters (OsIRT1, OsIRT2, and OsNRAMP1). In whole roots, these proteins are expressed in a coordinated manner with strong transcriptional induction in response to Fe deficiency. Radial transport of Fe to xylem and phloem is also mediated by the mugineic acid family phytosiderophores, as well as other chelators and their transporters, including Fe(II)-nicotianamine transporter (OsYSL2), phenolics efflux transporters (PEZ1 and PEZ2), and citrate efflux transporter (OsFRDL1). Among these, OsYSL2 is strongly induced under conditions of Fe deficiency. Both transcriptional induction and potential feedback repression mediate the expressional regulation of the genes involved in Fe uptake and translocation in response to Fe deficiency. The transcription factors IDEF1, IDEF2, and OsIRO2 are responsible for transcriptional induction, whereas the ubiquitin ligases OsHRZ1 and OsHRZ2, as well as the transcription factors OsIRO3 and OsbHLH133, are thought to mediate negative regulation. Furthermore, IDEF1 and OsHRZs bind Fe and other metals, and are therefore candidate Fe sensors. The interacting functions of these regulators are thought to fine tune the expression of proteins involved in Fe uptake and translocation.

Diurnal changes in the expression of genes that participate im phytosiderophore synthesis in rice

Abstract The roots of graminaceous plants secrete mugineic acid family phytosiderophores (MAs), allowing the acquisition of sparingly soluble iron (Fe) from the soil. In barley, the secretion of MAs occurs in a distinct diurnal rhythm. Rice also secretes deoxymugineic acid (DMA) in response to Fe deficiency, but it is unclear whether the secretion also occurs in a diurnal rhythm. A time course analysis with Northern blots was used to examine the expression of genes encoding enzymes in the MA biosynthetic pathway. The transcriptional levels of the NAS and NAAT genes fluctuated diurnally in Fe-deficient rice roots. These results suggest that, as in barley, the secretion of DMA in rice changes in a diurnal rhythm. Furthermore, the transcriptional levels of genes encoding translation initiation factor 4A2 (elF4A2), ras-related small GTP-binding protein (GTPase), and ADP-ribosylation factor 1 (ARF1), which fluctuate diurnally in barley and are thought to be involved in the diurnal changes in MA secretion, also changed diurnally. As these genes are involved in intracellular vesicular transport, it is possible that secretion of DMA in rice is under the control of vesicular transport, as in barley root cells.