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Featured researches published by Takanori Kobayashi.


Annual Review of Plant Biology | 2012

Iron Uptake, Translocation, and Regulation in Higher Plants

Takanori Kobayashi; Naoko K. Nishizawa

Iron is essential for the survival and proliferation of all plants. Higher plants have developed two distinct strategies to acquire iron, which is only slightly soluble, from the rhizosphere: the reduction strategy of nongraminaceous plants and the chelation strategy of graminaceous plants. Key molecular components-including transporters, enzymes, and chelators-have been clarified for both strategies, and many of these components are now thought to also function inside the plant to facilitate internal iron transport. Transporters for intracellular iron trafficking are also being clarified. A majority of genes encoding these components are transcriptionally regulated in response to iron availability. Recent research has uncovered central transcription factors, cis-acting elements, and molecular mechanisms regulating these genes. Manipulation of these molecular components has produced transgenic crops with enhanced tolerance to iron deficiency or with increased iron content in the edible parts.


Journal of Biological Chemistry | 2009

Rice OsYSL15 Is an Iron-regulated Iron(III)-Deoxymugineic Acid Transporter Expressed in the Roots and Is Essential for Iron Uptake in Early Growth of the Seedlings

Haruhiko Inoue; Takanori Kobayashi; Tomoko Nozoye; Michiko Takahashi; Yusuke Kakei; Kazumasa Suzuki; Mikio Nakazono; Hiromi Nakanishi; Satoshi Mori; Naoko K. Nishizawa

Graminaceous plants take up iron through YS1 (yellow stripe 1) and YS1-like (YSL) transporters using iron-chelating compounds known as mugineic acid family phytosiderophores. We examined the expression of 18 rice (Oryza sativa L.) YSL genes (OsYSL1-18) in the epidermis/exodermis, cortex, and stele of rice roots. Expression of OsYSL15 in root epidermis and stele was induced by iron deficiency and showed daily fluctuation. OsYSL15 restored a yeast mutant defective in iron uptake when supplied with iron(III)-deoxymugineic acid and transported iron(III)-deoxymugineic acid in Xenopus laevis oocytes. An OsYSL15-green fluorescent protein fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. OsYSL15 promoter-β-glucuronidase analysis revealed that OsYSL15 expression in roots was dominant in the epidermis/exodermis and phloem cells under conditions of iron deficiency and was detected only in phloem under iron sufficiency. These results strongly suggest that OsYSL15 is the dominant iron(III)-deoxymugineic acid transporter responsible for iron uptake from the rhizosphere and is also responsible for phloem transport of iron. OsYSL15 was also expressed in flowers, developing seeds, and in the embryonic scutellar epithelial cells during seed germination. OsYSL15 knockdown seedlings showed severe arrest in germination and early growth and were rescued by high iron supply. These results demonstrate that rice OsYSL15 plays a crucial role in iron homeostasis during the early stages of growth.


Journal of Biological Chemistry | 2011

Phytosiderophore Efflux Transporters Are Crucial for Iron Acquisition in Graminaceous Plants

Tomoko Nozoye; Seiji Nagasaka; Takanori Kobayashi; Michiko Takahashi; Yuki Sato; Yoko Sato; Nobuyuki Uozumi; Hiromi Nakanishi; Naoko K. Nishizawa

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released 14C-labeled deoxymugineic acid but not 14C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.


Journal of Biological Chemistry | 2008

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

Yuko Ogo; Takanori Kobayashi; Reiko Nakanishi Itai; Hiromi Nakanishi; Yusuke Kakei; Michiko Takahashi; Seiichi Toki; Satoshi Mori; Naoko K. Nishizawa

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.


Proceedings of the National Academy of Sciences of the United States of America | 2007

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

Takanori Kobayashi; Yuko Ogo; Reiko Nakanishi Itai; Hiromi Nakanishi; Michiko Takahashi; Satoshi Mori; Naoko K. Nishizawa

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.


Proceedings of the National Academy of Sciences of the United States of America | 2007

Mutational reconstructed ferric chelate reductase confers enhanced tolerance in rice to iron deficiency in calcareous soil

Yasuhiro Ishimaru; Suyeon Kim; Takashi Tsukamoto; Hiroyuki Oki; Takanori Kobayashi; Satoshi Watanabe; Shinpei Matsuhashi; Michiko Takahashi; Hiromi Nakanishi; Satoshi Mori; Naoko K. Nishizawa

Iron (Fe) deficiency is a worldwide agricultural problem on calcareous soils with low-Fe availability due to high soil pH. Rice plants use a well documented phytosiderophore-based system (Strategy II) to take up Fe from the soil and also possess a direct Fe2+ transport system. Rice plants are extremely susceptible to low-Fe supply, however, because of low phytosiderophore secretion and low Fe3+ reduction activity. A yeast Fe3+ chelate-reductase gene refre1/372, selected for better performance at high pH, was fused to the promoter of the Fe-regulated transporter, OsIRT1, and introduced into rice plants. The transgene was expressed in response to a low-Fe nutritional status in roots of transformants. Transgenic rice plants expressing the refre1/372 gene showed higher Fe3+ chelate-reductase activity and a higher Fe-uptake rate than vector controls under Fe-deficient conditions. Consequently, transgenic rice plants exhibited an enhanced tolerance to low-Fe availability and 7.9× the grain yield of nontransformed plants in calcareous soils. This report shows that enhancing the Fe3+ chelate-reductase activity of rice plants that normally have low endogenous levels confers resistance to Fe deficiency.


Planta | 2001

In vivo evidence that Ids3 from Hordeum vulgare encodes a dioxygenase that converts 2'-deoxymugineic acid to mugineic acid in transgenic rice

Takanori Kobayashi; Hiromi Nakanishi; Michiko Takahashi; Shinji Kawasaki; Naoko-Kishi Nishizawa; Satoshi Mori

Abstract. We proposed that an Fe-deficiency-induced gene, Ids3 (Iron deficiency specific clone no. 3), from barley (Hordeum vulgare L.) roots encodes a dioxygenase that catalyzes the hydroxylation step from 2′-deoxymugineic acid (DMA) to mugineic acid (MA). To prove this hypothesis, we introduced the Ids3 gene into rice (Oryza sativa L.), which lacks Ids3 homologues and secretes DMA, but not MA. Transgenic rice plants, carrying either Ids3 cDNA or a barley genomic DNA fragment (20 kb) containing Ids3, were obtained using Agrobacterium-mediated transformation. Ids3 cDNA under the control of the cauliflower mosaic virus 35S promoter was constitutively expressed in both the roots and the leaves of the transgenic rice, regardless of Fe nutrition status. In contrast, in the roots of transformants carrying a barley genomic fragment, transcripts of Ids3 were markedly increased in response to Fe deficiency. Slight expression of Ids3 was also observed in the leaves of the Fe-deficient plants. Western blot analysis confirmed the induction of Ids3 in response to Fe deficiency in the roots of the transformants carrying a genomic fragment. These expression patterns indicate that the 5′-flanking region of Ids3 works as a strong Fe-deficiency-inducible promoter in rice, as well as in barley. Both kinds of transgenic rice secreted MA in addition to DMA under Fe-deficient conditions, but wild-type rice secreted only DMA. This is in vivo evidence that IDS3 is the “MA synthase” that converts DMA to MA.


PLOS ONE | 2013

The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis).

Yoji Nakamura; Naobumi Sasaki; Masahiro Kobayashi; Nobuhiko Ojima; Motoshige Yasuike; Yuya Shigenobu; Masataka Satomi; Yoshiya Fukuma; Koji Shiwaku; Atsumi Tsujimoto; Takanori Kobayashi; Ichiro Nakayama; Fuminari Ito; Kazuhiro Nakajima; Motohiko Sano; Tokio Wada; Kiyoshi Inouye; Takashi Gojobori; Kazuho Ikeo

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.


Rice | 2009

Overexpression of the Barley Nicotianamine Synthase Gene HvNAS1 Increases Iron and Zinc Concentrations in Rice Grains

Hiroshi Masuda; Kanako Usuda; Takanori Kobayashi; Yasuhiro Ishimaru; Yusuke Kakei; Michiko Takahashi; Kyoko Higuchi; Hiromi Nakanishi; Satoshi Mori; Naoko K. Nishizawa

In humans, iron (Fe) and zinc (Zn) deficiencies result in major worldwide health problems. Transgenic technologies to produce Fe- and Zn-biofortified rice varieties offer a promising potential solution. Nicotianamine, the precursor of phytosiderophores, chelates Fe2+ and Zn2+ and plays an important role in transporting these metals to both vegetative and reproductive organs within the plant. Our objective was to increase Fe and Zn contents in rice grains by overexpressing the barley nicotianamine synthase gene HvNAS1. HvNAS1-overexpressing transgenic rice showed increased HvNAS1 expression and subsequent increases in endogenous nicotianamine and phytosiderophore content in shoots, roots, and seeds. Fe and Zn concentrations in polished T1 seeds from transgenic plants increased more than three and twofold, respectively; Fe and Zn concentrations also increased in both polished and brown T2 seeds. These results suggest that the overproduction of nicotianamine enhances the translocation of Fe and Zn into rice grains.


Plant Molecular Biology | 2009

OsYSL18 is a rice iron(III)–deoxymugineic acid transporter specifically expressed in reproductive organs and phloem of lamina joints

Takahiro Aoyama; Takanori Kobayashi; Michiko Takahashi; Seiji Nagasaka; Kanako Usuda; Yusuke Kakei; Yasuhiro Ishimaru; Hiromi Nakanishi; Satoshi Mori; Naoko K. Nishizawa

Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)–deoxymugineic acid, but not iron(II)–nicotianamine, zinc(II)–deoxymugineic acid, or zinc(II)–nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-β-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints.

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Naoko K. Nishizawa

Ishikawa Prefectural University

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May Sann Aung

Ishikawa Prefectural University

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Yoji Nakamura

National Institute of Genetics

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Motoshige Yasuike

Tokyo University of Marine Science and Technology

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