Report of Symposiumheld onJuly 14-15, 2009

ELUCIDATION OF RESISTANCE MECHANISMS TO ABIOTIC STRESSES AND THE APPLICATION FOR

MOLECULAR BREEDING

Report of Symposium held on

July 14-15, 2009

Institute of Radiation Breeding NIAS

Hitachi-Ohmiya, Ibaraki-ken Japan

I

The lecturers and the members of the Symposium Committee

II

Abe Tomoko RIKEN

Akemi Shimizu NIAS

Fujimori Masahiro Yamanashi Prefectural Daily Experiment Station

Fujino Kenji HOKUREN

Fujiwara Toru The University of Tokyo Fukaki Hidehiko Kobe University

Fukazawa Yoshitaka IBARAKI Prefectural Livestock Research Center Fukuda Akari National Agriculture and Food Research Organization

Fukuda Atsunori NIAS

Furukawa Koji Mukoyama Orchids Co. Ltd

Harada Kyuya NIAS

Hattori Kazumi Nagoya University

Hayashi Yoriko RIKEN

Hirochika Hirohiko NIAS

Horie Toshiya Fukui Prefecture

Inoue Eiichi Ibaraki University

Inukai Yoshaki Graduate School of Nagoya University

Ishige Teruo NIAS

Ishii Takuro NARO

Itoh Jun-ichi The University of Tokyo

Kanbe Takashi Niigata Agricultural Research Institute

Kazama Yusuke RIKEN

Kazuyuki Koide Society for Techno-Innovation of Agriculture, Forestry and Fisheries

Kobayashi Toru NIAS

Kobayashi Isao NIAS

Koike Setsuo National Agricultural Research Center Tohoku Region Konisho Kunihiko Nagano Nanshin Agricultural Experiment Station Kosuke Kazumasa IBARAKI Agricultural Center Plant-Biotechno1ogy Kuboyama Tsutomu Ibaraki University

Kurita Manabu Forest and Forest Products Research Institute Kusaba Makoto Hiroshima University

Mase Nobuko National Institute of Fruit Tree Science Masuda Tetsuo National Institute of Fruit Tree Science

Matsui Tsutomu Gifu University

Matsuo Youichi Saga Prefectural Fruit Tree Experiment Station Momnoki Yoshie Tokyo University of Agriculture

Morita Ryouhei NIAS

Muramatu Noboru NIAS

Nagamura Yoshiaki NIAS

Nagato Yasurou The University of Tokyo

Nakagawa Hitoshi National Institute of Agrobio1ogical Sciences

Nakagawa Mayu RIKEN

List of Participants

(48

GF Symposium)

III

N

Issay Japan Atomic Energy Agency

Nishihara Kiyoshi RIKEN

Nishimura Minoru NIAS

Nishio Takeshi Tohoku University

Nishizawa Naoko.K. Ishikawa Prefectural University

Nomoto Momoyo NIAS

Nozawa Shigeki Japan Atomic Energy Agency

Ohbu Sumie RIKEN

Ohmiya Yasunori Forest and Forest Products Research Institute Okazaki Keiichi Niigata University

Okumoto Yutaka Kyoto University

Sano Yoshio Hokkaido University

Sato Yutaka NIAS

Sekiguchi Fumihiko Japan Women’s University

Shibukawa Tomiko RIKEN

Sugimoto Kazuhiko NIAS

Takehisa Hinako NIAS

Takyu Toshio NIAS

Tamaki Katsutomo Hyogo Prefectural Institute for Agriculture Taniguchi Tohru Forestry and Forest Products Research Institute Tanisaka Takatoshi Kyoto University

Tsutsumi Nobuhiro University of Tokyo

Uga Yusaku NIAS

Ukai Yasuo

Yamanouchi Hiroaki NIAS

Yoshiaki Hitoshi Ishikawa Agricultural Research Center

IV

  Mutation breeding through chronic gamma-ray irradiation of growing plants in large irradiation facilities, such as our Gamma Field in Institute of Radiation Breeding, NIAS (Hitachi-Ohmiya, Ibaraki, Japan) has been expanding in Asia. In 2009, a Gamma Greenhouse was established for the facilitation of mutation breeding through chronic gamma-ray irradiation to growing plants and in vitro materials in Agrotechnology & Biosciences Division of Malaysian Nuclear Agency (Bangi 43000 Kajang, Selangor, Malaysia). A “Mutation induction using chronic irradiation with gamma greenhouse” Training/Workshop was conducted for the opening of the facility with Prof.

Siranut Lamseejan of Kasetsart University and Dr. Hitoshi Nakagawa, IRB, NIAS with lecturers provided on 3-7 August 2009 for the promotion of this technology at the new facility. Although the history of mutation breeding is more than 50 years old and has been useful for the improvement of crops, the differences in the induced mutations between acute and chronic irradiation are not well defied or understood. The application of chronic irradiation to growing plants in the field or controlled greenhouse will be useful to elucidate the point and provide an outlet for the development of new crop varieties.

  On 12-15 August 2008, “the FAO/IAEA International Symposium on Induced Mutation in Plant” was held for celebrating the 80

anniversary of mutation induction in plant in Vienna, Austria. The symposium was organized by IAEA and FAO through the Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture cooperated with Bhabha Atomic Research Center, Chinese Society of Agricultural Biotechnology, European Association for Research on Plant Breeding, Indian Society of Genetics and Plant Breeding, and National Institute of Agrobiological Sciences, Japan. More than 400 persons from 83 countries attended the symposium. The proceedings are published as a book, “Induced Plant Mutations in the Genomic Era” edited by Q. Y. Shu from Food and Agriculture Organization of the United Nations, Rome, in 2009. The 458 page of proceedings provide details regarding accomplishments, progress and the future directions of mutation breeding.

   The 1

Gamma Field Symposium was held in 1962 at Conference Room in the Institute of Radiation Breeding for exchanging information and discussions among scientists in national agricultural experiment stations and institutes of Ministry of Agriculture and Forestry, national universities and institutes of Ministry of Education, and seed companies in this new research area of mutation breeding, and for providing a seminar to students of the universities. During its 48-year history, the symposium committee has selected various themes related to mutation and breeding, and has invited leading scientists with expertise in these areas as lecturers to provide results of their research on a wide variety of related topics.

  The 48

Gamma Field Symposium entitled “Elucidation of resistance mechanisms to abiotic stresses and the application for molecular breeding” was held on July 15-16, 2009 in Mito, Ibaraki, Japan. The keynote address, Genes involved in ion-acquisition and their application for developing new crops” was presented by Prof. N. K.

Nishizawa, Professor of Ishikawa Prefectural University. Prof. Nishioka was a professor of Graduate School of Agricultural and Life Science, The Univerity of Tokyo, and regarded as one of the most renowned scientists regarding mechanism of abiotic stresses in plants. Seven lecturers were also invited to present results of their research results:

Prof. T. Fujiwara (The University of Tokyo: Molecular mechanisms of boron transport in plants and generation of plants tolerant to boron stress); Prof. H. Fukaki (Kobe University: Genetic regulation of lateral root development in Arabidopsis -The role of auxin signaling-); Prof. Y. Inukai (Nagoya University: Genetic improvement of root system formation for adaptation to soil moisture fluctuation stress in rice); Prof. T. Matsui (Gifu University: Heat- induced floret sterility in rice: Mechanisms of occurrence and tolerance); Dr. K. Sugimoto (National Institute of Agrobiological Sciences: Genetic control of seed dormancy in rice); Prof. Y. S. Momonoki (Tokyo University of

Forward

V

Agriculture: Characterization of the plant acetylcholine-mediated system); and Dr. I. Narumi (Japan Atomic Energy Agency: Survival strategy of a radioresistant bacterium: a review).

   This publication includes the contributed papers from these invited lecturers and subsequent questions and discussions (in Japanese) addressed to the invitees following their presentations during the symposium.

   It is our sincere hope that the series of Gamma Field Symposia, including this issue, will assist plant breeders and researchers to realize the contribution that mutation breeding has made to the plant sciences.

   The most recent and previous volumes of Gamma Field Symposia series have been placed on line and can be accessed at the link http://www.nias.affrc.go.jp/eng/gfs/index.html .

   We express our sincere thanks to the lectures, chairpersons and attendees

Symposium Committee Hitoshi Nakagawa, Chairperson Yasuro Nagato

Hirohiko Hirochika

Tetsuo Masuda

Yoshio Sano

Takatoshi Tanisaka

Nobuhiro Tsutsumi

Minoru Nishimura, Secretary

Noboru Muramatsu, Editor

VI

Special lecture Chairperson: N. T

Genes Involved in Iron Acquisition and Their Application for Developing New Crops ……… N. K. N

Session Ⅰ

Chairperson: T. M

Molecular Mechanisms of Boron Transport in Plants and Generation of Plants Tolerant to Boron Stress.

  ……… T. F

Session Ⅱ Chairperson: Y.N

Genetic Regulation of Lateral Root Development in Arabidopsis

  – The Role of Auxin Signaling – ……… H. F

Session Ⅲ Chairperson: M. K

Genetic Improvement of Root System Formation for Adaptation to Soil Moisture Fluctuation Stress in Rice.

  ………Y. I

Session Ⅳ Chairperson: T. T

Heat-induced Floret Sterility in Rice: Mechanisms of Occurrence and Tolerance. ……… T. M

Session Ⅴ

Chairperson: H. H

Genetic Control of Seed Dormancy in Rice ……… K. S

Session VI

Chairperson: H. N

Characterization of the Plant Acetylcholine-Mediated System ……… Y. S. M

Session VII

Chairperson: T. A

Survival Strategy of a Radioresistant Bacterium ……… I. N

Session VIII Chairperson: Y. S

General Discussion Closing address: Y. N

PROGRAM

Opening address: H. N

Congratulatory address: M. I

VII CONTENTS

T. K

Genes Involved in Iron Acquisition and Their Application for

H. N

Developing New Crops ……… 1 N. K. N

T. F

Molecular Mechanisms of Boron Transport in Plants and Generation of

Plants Tolerant to Boron Stress. ……… 17 H. F

Genetic Regulation of Lateral Root Development in Arabidopsis

– The Role of Auxin Signaling – ……… 27 Y. I

Genetic Improvement of Root System Formation for Adaptation to

Soil Moisture Fluctuation Stress in Rice. ……… 35 T. M

Heat-induced Floret Sterility in Rice: Mechanisms of Occurrence and Tolerance. …… 43 N. L. M

E. R

X. T

M. Y

K. S

Genetic Control of Seed Dormancy in Rice ……… 53 S. M

M. Y

Y. S. M

Characterization of the Plant Acetylcholine-Mediated System ……… 61 K. Y

I. N

Survival Strategy of a Radioresistant Bacterium: a Review ……… 69

General Discussion (in Japanese) ……… 77

1

Gamma Field Symposia, No. 48, 2009 Institure of Radiation Breeding NIAS, Japan

Genes Involved in Iron Acquisition and Their Application for Developing New Crops

Takanori K

, Hiromi N

and Naoko K. N

1

Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

2

Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-machi, Ishikawa 921-8836, Japan

Abstract

Iron deficiency is a major cause of reduced crop yields worldwide, particularly in calcareous soils. Un- like barley, rice is highly susceptible to iron deficiency because of a low capacity to secrete phytosiderophores in the mugineic acid (MA) family, which are iron chela- tors secreted by graminaceous plants. In this paper, we present an approach to the generation of transgenic rice lines exhibiting increased tolerance to iron deficiency, along with the results of field trials with these lines.

Cloning barley genes that encode biosynthetic enzymes for MAs enabled us to produce transgenic rice plants that contained these genes. We tested three transgenic lines possessing barley genomic fragments responsible for biosynthesis of MAs in a paddy field experiment on calcareous soil, and demonstrated tolerance of these lines to low iron availability. We also applied new ap- proaches to generate iron-deficiency-tolerant rice lines, including the introduction of an engineered ferric-che- late reductase gene and manipulation of transcription- factor genes that regulate the iron-deficiency response.

1. Introduction

Iron (Fe) is essential for most living organisms, including plants. Although it is abundant in mineral soils, Fe is sparingly soluble under aerobic conditions at high soil pH, especially in calcareous soils, which ac- count for about 30% of the world’s cultivated soils. Fe deficiency is a widespread agricultural problem that re-

duces plant growth and crop yields (M

1995;

M

1999). To take up and utilize Fe from the rhizo- sphere, higher plants have evolved two major strategies (M

et al. 1986): reduction (Strategy I) and chelation (Strategy II). The mechanism of Strategy I, which is utilized by non-graminaceous plants, includes the production of ferric-chelate reductase to reduce Fe at the root surface to the more soluble ferrous (Fe[II]) form, and transport the ferrous ions generated by this process across the root plasma membrane. In contrast, the mechanism of Strategy II, which is specific to gram- inaceous plants, is mediated by natural Fe chelators, the mugineic acid (MA) family of phytosiderophores.

Graminaceous plants synthesize and secrete MAs from their roots to solubilize Fe(III) in the rhizosphere (T

AGI

1976), and the resulting Fe(III)–MA complexes are taken up by roots through a specific transporter in the plasma membrane (C

et al. 2001; T

1976).

In calcareous soils, Strategy II is more efficient than Strategy I (M

1995). Tolerance of Fe deficiency differs among graminaceous plants and is thought to depend on the amount and kinds of MAs that they secrete. Rice (Oryza sativa L.), sorghum (Sorghum bicolor L.), and maize (Zea mays L.) secrete only small amounts of 2’-deoxymugineic acid (DMA) among the MAs, and thus are susceptible to low Fe availability.

In contrast, barley (Hordeum vulgare L.) secretes large

amounts of compounds in the MA family, including

MA, 3-epihydroxy-2’-deoxymugineic acid (epiHDMA)

and 3-epihydroxymugineic acid (epiHMA), in addition

to DMA, under Fe deficiency; therefore, barley is more

2

tolerant of Fe deficiency than other graminaceous plants (M

et al. 1999; M

and N

1987; R

and M

1986; T

1976).

We therefore hypothesized that introducing the barley genes responsible for biosynthesis of MAs into rice would lead to enhanced production of MAs and tolerance of Fe deficiency in calcareous soils. We suc- cessfully produced various transgenic rice lines with enhanced tolerance of low Fe availability by introduc- ing barley biosynthesis genes for MAs. Using selected lines, we carried out field trials in a calcareous soil that demonstrated the tolerance of these lines to low Fe availability. We also produced Fe-deficiency-toler- ant rice lines using two other strategies: introduction of a reconstructed ferric-chelate reductase gene and manipulation of transcription-factor genes that control the expression of Fe-deficiency-induced genes. Based on these results, we discuss future perspectives on gen- erating agriculturally beneficial transformants that are also acceptable to the public.

2. Generation of Fe-deficiency-tolerant transgenic rice by introducing barley biosynthesis genes for MAs 2.1. Identification of genes responsible for biosyn-

thesis of MAs

The biosynthetic pathway for MAs (Fig. 1) has been clarified through extensive biochemical and phys- iological studies (K

et al. 1988; M

and N

1993; M

et al. 1999; M

and N

1987;

S

et al. 1990). Methionine is the precursor of MAs (M

and N

1987) and is adenosylated by S-adenosylmethionine (SAM) synthetase (SAMS).

Nicotianamine synthase (NAS) catalyzes the trimeriza- tion of SAM into nicotianamine (NA) (H

et al.

1994). All higher plants, including non-graminaceous plants, have a biosynthetic pathway that lets them syn- thesize NA (L

et al. 1999; N

and N

1976), which serves as a common metal chelator in- volved in the internal transport of various micronutri- ents, including Fe and zinc (Zn) (H

and S

Fig. 1. Biosynthetic pathways of phytosiderophores in the mugineic acid (MA) family.

SAMS, S-adenosylmethionine synthetase; NAS, nicotianamine synthase; NAAT, nicotianamine aminotransferase; DMAS, deoxymugineic acid synthase; IDS2, iron-deficiency-specific clone no. 2; IDS3, iron-deficiency-specific clone no. 3;

DMA, 2’-deoxymugineic acid; MA, mugineic acid; HMA, 3-hydroxymugineic

acid; epiHDMA, 3-epihydroxy-2’-deoxymugineic acid; epiHMA, 3-epihydroxy-

mugineic acid.

3

2003; T

et al. 2003). NA aminotransferase

(NAAT) catalyzes the first step specific to gramina- ceous plants: transamination of NA to produce the 3’’- oxo intermediate (K

et al. 1995; S

et al. 1990). DMA synthase (DMAS) subsequently reduc- es the 3’’-oxo form into DMA (B

et al. 2006). All MAs share the same biosynthetic pathway from me- thionine to DMA, which is then hydroxylated to form other MAs in some species, including barley.

Attempts to isolate the genes responsible for bio- synthesis of MAs have included “direct” approaches via enzyme purification and “indirect” approaches through screening the genes and proteins specifically induced in Fe-deficient roots. The former approach has been applied to NAS and NAAT. NAS genes were first isolated from barley (HvNAS1-7) through the es- tablishment of an NAS activity assay (H

et al.

1994) and enzyme purification from Fe-deficient barley roots (H

et al. 1999). Two barley NAAT genes, HvNAAT-A and HvNAAT-B, were also cloned through the establishment of an enzyme activity assay (O

et al. 1993) and enzyme purification (K

et al.

1995; T

et al. 1999). Expression of HvNAS1, HvNAAT-A, and HvNAAT-B is strongly induced by Fe deficiency and occurs almost exclusively in the roots (H

et al. 1999; T

et al. 1999), suggest- ing direct involvement of these genes in the biosynthe- sis of MAs to increase the acquisition of Fe from the rhizosphere. Quantification of NAS and NAAT enzyme activities in Fe-deficient roots of various graminaceous species have revealed that NAS and NAAT activities are positively correlated with both the amounts of MAs secreted and with Fe-deficiency tolerance (H

et al. 1996; K

et al. 1994).

In the indirect approach, the differential hybrid- ization method was applied with mRNA from Fe-de- ficient and Fe-sufficient barley roots. We cloned IDS (iron-deficiency specific) genes specifically expressed in Fe-deficient barley roots (N

et al. 1993;

O

et al. 1991, 1994). Among these, IDS2 and IDS3 are homologous to 2-oxoglutarate-dependent di- oxygenases, suggesting their possible involvement in the hydroxylation of MAs. By interspecies correlation

between the expression of IDS2 and IDS3 and the ca- pacity to secrete hydroxylated MAs, we deduced that IDS3 is the enzyme that hydroxylates the C-2’ positions of DMA and epiHDMA, whereas IDS2 hydroxylates the C-3 positions of DMA and MA (Fig. 1; N

SHI

et al. 2000). IDS3 was further confirmed to be MA synthase by introducing the barley IDS3 gene into rice (K

et al. 2001): transgenic rice plants secreted MA in addition to DMA, whereas non-transformants secreted only DMA.

We also compared proteins in Fe-sufficient and Fe-deficient barley roots using two-dimensional poly- acrylamide gel electrophoresis. Peptide sequencing of the proteins produced under Fe deficiency revealed that formate dehydrogenase (FDH) and adenine phosphori- bosyltransferase (APRT), as well as the IDS3 protein, were produced in Fe-deficient roots (K. S

et al.

1998). The corresponding genes, HvFDH and HvAPT, were subsequently cloned (I

et al. 2000; S

et al. 1998). Both FDH and APRT are thought to function in scavenging the by-products (formate and adenine) that are released during the methionine cycle (M

1999), thus supporting the production of MAs. Indeed, the methionine cycle works vigorously in roots to meet the increased demand for methionine to support the synthesis of MAs (M

et al. 1995). We also applied a revised differential-hybridization screening, and identi- fied iron-deficiency-induced (IDI) genes in barley roots (Y

et al. 2000a, 2000b, 2002). IDI1 and IDI2 putatively encode enzymes that catalyze steps in the methionine cycle (K

et al. 2005; M. S

et al. 2006).

Recent application of microarray techniques re-

confirmed the induction of the abovementioned genes

involved in biosynthesis of MAs in Fe-deficient barley

roots (N

et al. 2002; M. S

et al. 2006). The

microarray approach also resulted in cloning of DMAS

genes from rice (OsDMAS1), barley (HvDMAS1), wheat

(Triticum aestivum L.; TaDMAS1), and maize (ZmD-

MAS1). All of the corresponding encoded proteins were

confirmed to possess the reductase activity required to

produce DMA (B

et al. 2006).

4

2.2. Introduction of barley genes responsible for bio- synthesis of MAs into rice

To produce transgenic rice plants with enhanced tolerance of Fe deficiency by increasing their capacity for the production of MAs, we introduced barley Hv- NAS1, HvNAAT-A, HvNAAT-B, or IDS3 genes, or some combination of these genes, using either their genomic fragments or the IDS3 gene promoter to confer induc- ibility under Fe deficiency. To introduce barley genomic fragments, we utilized the pBIGRZ1 vector (A

et al. 1997), which was developed as a modified binary vector capable of transferring large DNA fragments into the rice genome. Rice cultivar Tsukinohikari was subjected to Agrobacterium-mediated transformation (H

et al. 1994). The transformants included lines in which the following genes or gene fragments were introduced: (a) a 13.5-kb genome fragment of the Hv- NAS1 gene (H

et al. 2001), designated “gNAS1”;

(b) an 11-kb genome fragment of HvNAAT in which the HvNAAT-A and HvNAAT-B genes are present in tandem (Takahashi et al. 2001), designated “gNAAT”; (c) a 20- kb genome fragment of the IDS3 gene (K

et al. 2001), designated “gIDS3”; (d) a 7.6-kb genome fragment of HvNAS1 plus an 11-kb genome fragment containing the HvNAAT-A and HvNAAT-B genes, des- ignated “gNAS1-gNAAT”; (e) an HvAPT cDNA frag- ment fused downstream of the 2.2-kb IDS3 promoter;

(f) HvNAS1, HvNAAT-A, and HvAPT cDNA fragments, each fused downstream of the 2.2-kb IDS3 promoter;

and (g) an IDS3 cDNA fragment fused downstream of the cauliflower mosaic virus (CaMV) 35S promoter (K

et al. 2001). Expression analysis revealed that Fe-deficiency-induced expression was strongly conferred by genome fragments of the HvNAS1, Hv- NAAT-A, HvNAAT-B, or IDS3 genes (H

et al.

2001; K

et al. 2001; T

et al. 2001), confirming the potency of the barley promoter elements included in the genome fragments and their ability to drive Fe-deficiency-induced expression in rice plants.

Although the barley promoters cause induction of gene expression almost exclusively in Fe-deficient roots in their native barley, they induced moderate expression in Fe-deficient leaves and prominent expression in Fe-

deficient roots when introduced into rice.

To examine whether the transformants have en- hanced tolerance of low Fe availability, the plants were cultured in pots filled with calcareous soils (pH 8.5 to 9.0; T

et al. 2001) under controlled condi- tions in a greenhouse. Of the 36 gNAAT lines that we evaluated, 10 showed remarkable tolerance of calcare- ous soils (T

et al. 2001). Non-transformants exhibited reduced growth and severe leaf chlorosis caused by Fe deficiency, whereas the gNAAT lines had greener and larger shoots. At harvest, the gNAAT lines possessed 4.2 and 4.1 times the shoot dry weight and grain yield per pot that we found in the non-transfor- mants (T

et al. 2001). We also examined tol- erance of calcareous soils for the other transformants.

We found rice lines that showed some tolerance of cal- careous soils among lines containing any of transgenes (a) through (g). In these lines, increased amounts or kinds of secreted MAs appear to have contributed to enhanced Fe availability under Fe-limiting conditions.

3. Field trials of Fe-deficiency-tolerant rice lines A calcareous subsoil from Toyama Prefecture con- taining fossil shells (pH ~9.2; M

et al. 2004) was used to establish a paddy field in the quarantine area of the Field Science Center of Tohoku University (Osaki, Miyagi, Japan; 38°44’N; 140°45’E). The paddy field in the first-year experiment was 7 m long by 14 m wide, and had soils 0.5 m deep, with the external ridges completely covered with a vinyl sheet to avoid con- tamination from the surrounding Andosol at the site.

The first-year experiment was conducted from April to October 2005, using the six transformant lines (a) through (f) and a non-transformant cultivar (Tsukinohi- kari). The following year, from April to October 2006, the second-year experiment was performed using the three most promising lines: gNAS1 (a), gIDS3 (c), and gNAS1–gNAAT (d). Experimental procedures for the second-year experiment were described by M. S

et al. (2008). The paddy field in the second-year ex-

periment was 6 m long by 4 m wide (Fig. 2a), and the

experimental plots were arranged in a completely ran-

5

domized design (Fig. 2b) that included the three trans-

genic rice lines (gNAS1, gIDS3, and gNAS1–gNAAT) and the non-transformant (NT). Germinated seeds were grown for 45 days in a greenhouse, and seedlings were then transplanted (three per hill) into the calcareous paddy field.

Sixteen days after transplanting (DAT), chlorosis and growth retardation began to appear. By 42 DAT, the three transgenic rice lines were clearly superior to the NT (Fig. 2a) both in leaf color and in growth, although differences in performance were also observed within individual plots. Using gIDS3 as an example, we saw no evident difference from the NT on 16 DAT (Fig.

3a); chlorotic symptoms appeared in the NT but not in gIDS3 at 30 DAT (Fig. 3b). The clearest difference be- tween gIDS3 and the NT was evident at 42 DAT (Fig.

3c). One week later (50 DAT), leaf chlorosis began to disappear, especially in NT plants close to the plot

boundary that were adjacent to the transgenic gIDS3 rice plants (Fig. 3d), which suggests that the NT plants may have benefited from MAs secreted by the trans- genic rice.

From 16 to 42 DAT, plant height and the SPAD value (leaf color) of the three transformant lines were higher than those of the NT. In addition, the number of tillers per plant was higher in gIDS3 than in the other lines. By 42 DAT, however, all lines had about 15 til- lers per plant. After 42 DAT, when soil redox poten- tial (E

) fell below 0 mV, all plant lines recovered their leaf color, and consequently, the SPAD value of the NT plants increased to levels similar to those in the trans- formants. The decrease in soil E

with time is thought to have resulted in the absorption of ferrous ions via the ferrous transporter OsIRT1 (see section 4.1 for details;

I

et al. 2006).

At the time of grain harvesting, the number of

Fig. 2. (a) Photograph (42 days after transplanting) of the rice lines tested in a paddy field in the quarantine area of the Field Science Center of Tohoku University (Osaki, Miyagi, Japan) and (b) layout of the field experi- ment. Each population contained five 1.2-m-long rows of rice with 20 cm between rows and 15 cm between hills. The box in the upper left in- dicates the two plots that were photographed on several occasions (Fig.

3). NT, non-transformant. Original figure: S

et al. (2008).

6

grains, 1000-grain weights, and the grain yield of gNAS1 were higher than those of the NT plants and the other lines. Plant height and the proportion of fully ma- tured grains showed no significant difference among the lines. The timing of the decrease in soil E

might account for the relatively small differences in grain parameters between the transformants and NT, despite the clearly inferior performance of NT plants during early growth.

Indeed, in a prior experiment, NT seedlings grown in the same calcareous paddy field showed severe chloro- sis, and many seedlings died in the early stages before E

fell below 0 mV (M

et al. 2004). Therefore, it is crucial for rice in calcareous paddy fields to survive the early stages of growth, when enhanced production

of MAs greatly supports Fe acquisition.

Interestingly, the concentrations of Fe and Zn in the rice grains of gIDS3 were significantly higher than those in NT plants and the other lines, suggesting that MA synthesized by IDS3 contributed not only to im- proved Fe uptake from the soil but also to increased translocation to the grains.

Our field trial of the transformants therefore dem- onstrated that a transgenic approach to increase the tol- erance of rice to low Fe availability is a practical way to improve agricultural productivity in calcareous paddy soils.

4. Production of other transgenic rice plants toler- ant of Fe deficiency

4.1. Introducing an engineered ferric-chelate reduc- tase gene into rice

In Strategy I plants, Fe uptake from the rhizo- sphere is mediated by ferrous ion transporters. E

et al. (1996) isolated the Arabidopsis IRT1 gene, which is the dominant ferrous Fe transporter in the Fe-up- take process (V

et al. 2002). Rice, in spite of be- ing a Strategy II plant, possesses homologs of the Arabidopsis IRT1 gene, namely OsIRT1 and OsIRT2, and the ferrous transport capacity of these genes was demonstrated by means of functional complementation in yeast (B

et al. 2002; I

et al. 2006).

OsIRT1 expression is strongly induced in Fe-deficient roots, and OsIRT2 is expressed similarly but at lower levels. Promoter-β-glucuronidase (GUS) analysis indi- cated that OsIRT1 is mainly expressed in the epidermis, exodermis, and inner layer of the cortex in Fe-deficient roots, as well as in shoot companion cells. Moreover, an analysis using a positron-emitting tracer-imaging system (PETIS) revealed that rice is able to take up both Fe(III)-DMA and Fe

. Thus, rice plants possess a system other than Strategy II for Fe uptake, which is based on the secretion of MAs (I

et al. 2006).

In contrast to their ferrous transporting ability, Fe-de- ficient rice roots do not show increased ferric-chelate reductase activity (I

et al. 2006), which is a hallmark of the Strategy I response.

Fig. 3. Visual comparison between gIDS3 (left) and NT

(right) plants from 16 to 50 DAT, as illustrated in

Figure 2 but photographed from the opposite di-

rection. (a) 16 DAT, (b) 30 DAT, (c) 42 DAT, and

(d) 50 DAT. Original figure: S

et al. (2008).

7

The ability to take up ferrous ions directly using

OsIRT1, without reducing ferric chelates, seems to be a consequence of rice’s adaptation to waterlogged soils, in which the concentration of soluble ferrous Fe increases as the soil E

decreases (I

et al. 2006; S

et al. 2008). Because of the presence of OsIRT1, severe Fe deficiency is relatively rare in irrigated rice systems.

Nevertheless, rice plants grown in calcareous soils ex- hibit Fe-deficiency symptoms even under waterlogged conditions (as noted previously) because of their inabil- ity to increase ferric-chelate reductase production and their low capacity to synthesize MAs. Therefore, we hypothesized that introducing a ferric-chelate reductase gene into rice would enhance its Fe-deficiency toler- ance by creating a complete Strategy I system in addi- tion to rice’s endogenous Strategy II.

To ensure functional expression in the rice plants, we modified and completely reconstructed the yeast ferric reductase gene, FRE1, to produce refre1 (recon- structed FRE1; O

et al. 1999). Since ferric-chelate reductase activity is inhibited by high pH, we then screened plants to detect reductases with improved en- zymatic activity at high pH (O

et al. 2004). Through screening of randomly mutagenized refre1 derivatives, we obtained a variant that we designated refre1/372, which encoded a protein that maintained strong reduc- tase activity at pH 8 to 9. Transgenic tobacco (Nicotiana

tabacum L.) plants with the introduced refre1/372 gene under control of the CaMV 35S promoter exhibited en- hanced ferric-chelate reductase activity in the roots and better growth when grown in calcareous soils (O

et al. 2004).

Another concern in relation to the introduction of exogenous reductase genes into rice was the choice of an appropriate promoter. V

et al. (2004) in- troduced the Arabidopsis ferric-chelate reductase gene FRO2 with its own promoter, but observed no transgene mRNA production. In Arabidopsis, the production of ferric-chelate reductase FRO2 and ferrous transporter IRT1 is similarly and coordinately regulated at tran- scriptional and post-transcriptional levels (C

et al. 2003; V

et al. 2003). Therefore, we chose the promoter of the rice ferrous transporter gene (OsIRT1) to drive the exogenous ferric-chelate reductase gene re- fre1/372 (I

et al. 2007).

Transgenic rice plants with the introduced OsIRT1 promoter, refre1/372, successfully produced ferric-che- late reductase and activity in Fe-deficient roots, leading to higher Fe uptake than by vector controls, as revealed by a PETIS analysis. The transformants exhibited en- hanced tolerance of low Fe availability in both hydro- ponic culture (data not shown) and calcareous soil (Fig.

4a). When grown in calcareous soil until harvest, the transformants had 7.9 times the grain yield of the vector

Fig. 4. Tolerance of Fe deficiency in transformants with the introduced OsIRT1 promoter-refre1/372 grown

in calcareous soil. (a) Transformants (TF, left) and vector controls (V, center) after 4 weeks of growth

in a calcareous soil, and vector controls (V, right) in a bonsol (a normal cultivated soil). (b) Transfor-

mants (TF, left) and vector control (V, right) after 17 weeks of growth in calcareous soil. (c) Grain

yield after cultivation for 17 weeks in calcareous soil. Original figure: I

et al. (2007).

8

controls (Fig. 4b, c; I

et al. 2007), demonstrat- ing that creating a complete Strategy I system in rice by enhancing ferric-chelate reductase activity is extremely effective in improving Fe-deficiency tolerance.

4.2. Manipulating the transcription factors that reg- ulate the Fe-deficiency response

The abovementioned studies have shown that in- troduction of only a single gene or a few genes can ef- fectively confer Fe-deficiency tolerance if one or more appropriate promoters and genes are utilized. How- ever, further enhancement of Fe availability might be achieved by engineering multiple genes to operate in a coordinated manner. The genetic enhancement of a wide range of related genes requires manipulation of basal regulatory systems, including transcription fac- tors. Therefore, we also aimed to clarify the regulatory mechanisms that control the Fe-deficiency response in graminaceous plants.

Under low Fe availability, graminaceous plants in- duce the expression of various genes, many of which are involved in Fe acquisition and utilization (B

et al. 2006; K

and N

2008; K

BAYASHI

et al. 2005; M

1999; N

et al. 2002).

Despite the number of Fe-deficiency-inducible genes that have been isolated, little is known about the regula- tion of gene expression in response to Fe deficiency.

Therefore, we applied a stepwise strategy to identify the molecular components that regulate the expression of Fe-deficiency-responsive genes: the establishment of a promoter assay system, identification of cis-acting elements, and identification of trans-acting factors that interact with the cis-acting elements.

We introduced the promoter region of the barley IDS2 gene connected to the GUS gene as a reporter into tobacco plants (Y

et al. 2003). Transgenic tobacco plants exhibited GUS expression in Fe-defi- cient roots, basically reflecting the regulation pattern in the gene’s native barley. Precise deletion and mutation analyses using numerous lines of transgenic tobacco identified two novel Fe-deficiency-responsive cis-acting elements, IDE1 and IDE2 (iron-deficiency-responsive elements 1 and 2; K

et al. 2003); these are the

first identified elements that are related to micronutri- ent deficiencies in plants. IDE1 and IDE2 synergisti- cally induce the expression of Fe-deficiency-responsive genes in tobacco roots. When introduced into rice, the pair IDE1 and IDE2 can induce the expression of Fe- deficiency-responsive genes in both roots and leaves (K

et al. 2004). Sequences similar to IDE1 or IDE2 have been found in various Fe-deficiency-induc- ible promoters in barley, rice, tobacco, and Arabidop- sis (D

et al. 2005; K

et al. 2003, 2005).

This suggests that gene regulation mechanisms involv- ing IDEs are not only conserved among graminaceous (Strategy II) plants but are also functional in non-gram- inaceous (Strategy I) plants.

Next, we searched for transcription factors that interact with IDEs. We recently successfully identi- fied two rice transcription factors, IDEF1 (IDE-binding factor 1) and IDEF2, which specifically bind to IDE1 and IDE2, respectively (K

et al. 2007; O

et al. 2008). IDEF1 and IDEF2 belong to uncharacter- ized branches of the plant-specific transcription factor families ABI3/VP1 and NAC, respectively, and exhibit novel sequence-recognition properties. IDEF1 rec- ognizes the CATGC sequence within IDE1, whereas IDEF2 predominantly recognizes CA[A/C]G[T/C][T/

C/A][T/C/A] within IDE2 as the core binding site. Both IDEF1 and IDEF2 are constitutively expressed in rice roots and leaves.

In an attempt to improve Fe-deficiency tolerance by modulating IDEF1 expression, we introduced IDEF1 cDNA fused to either the constitutive CaMV 35S pro- moter or the Fe-deficiency-inducible IDS2 promoter.

Transgenic rice seedlings with the introduced CaMV 35S promoter–IDEF1 construct showed severe growth retardation during early growth, whereas those carrying the IDS2 promoter–IDEF1 construct showed healthy growth. Notably, the IDS2 promoter–IDEF1 transfor- mants exhibited slower progression of leaf chlorosis in Fe-free hydroponic culture, and also showed better growth when germinated on calcareous soil (Fig. 5; K

et al. 2007).

To clarify the molecular mechanisms that regulate

Fe acquisition, we also characterized the Fe-deficiency-

9

induced transcription factors. Microarray analyses re- vealed the upregulation of several transcription factor genes in barley and rice (N

et al. 2002; O

et al.

2006), among which a bHLH transcription factor gene, IRO2, is of particular interest because of its pronounced transcriptional upregulation by Fe deficiency in shoots and roots of barley and rice (O

et al. 2006). The core sequence for OsIRO2 binding was determined to be CACGTGG (O

et al. 2006).

We produced transgenic rice plants with enhanced or repressed OsIRO2 expression by introducing the CaMV 35S–OsIRO2 cassette or using the RNA inter- ference (RNAi) technique (O

et al. 2007). In Fe- deficient hydroponic culture, OsIRO2-overexpressing lines showed enhanced secretion of MAs and slightly better growth compared to non-transformants, where- as OsIRO2-repressed lines showed lower secretion of MAs and hypersensitivity to Fe deficiency. Microarray and northern blot analyses revealed that the expres- sion level of OsIRO2 was positively related to that of various Fe-deficiency-induced genes in roots, including those responsible for biosynthesis of MAs (OsNAS1, OsNAS2, OsNAAT1, OsDMAS1, and various genes in- volved in the methionine cycle) and Fe(III)-MA uptake (OsYSL15). OsIRO2 also affects the expression of some Fe-deficiency-inducible transcription factor genes that possess OsIRO2-binding core sequences in their pro- moter regions (O

et al. 2007). Importantly, OsIRO2 itself possesses multiple IDEF1-binding core sequenc- es in its promoter region and is positively regulated by

IDEF1 (K

et al. 2007). Based on these results, we have proposed a sequential link in the Fe-deficiency response that involves IDEF1, IDEF2, OsIRO2, and downstream Fe-deficiency-inducible transcription fac- tors (Fig. 6; K

et al. 2007; O

et al. 2007, 2008).

In contrast to the growth retardation observed in the CaMV 35S promoter–IDEF1 transformants, the CaMV 35S promoter–OsIRO2 transformants were healthy, and Fig. 5. Tolerance of Fe deficiency in seedlings with the

introduced IDS2 promoter–IDEF1 construct ger- minated in a calcareous soil (lines 9, 12, and 13) compared to non-transformant (NT) plants 17 days after sowing. Original figure: K

et al. (2007).

Fig. 6. Proposed regulatory network for the induction of Fe-deficiency-responsive genes

and OsIRO2. Under Fe-deficiency conditions, IDEF1 and IDEF2 transactivate the expression of Fe-deficiency-responsive genes by binding to the IDE1-like and IDE2-like elements, respectively (K

et al. 2007; O

et al. 2008). OsIRO2 expression, which is induced by Fe-deficiency and is positively regulated by IDEF1, produces OsIRO2 that binds to CACGTGG elements to ac- tivate another subset of Fe-deficiency-responsive genes, including two transcription factor genes:

OsNAC4 and the gene that contains an AP2 do-

main. These transcription factors may then regu- late Fe-deficiency-responsive genes that lack IDEs and CACGTGG in their promoter regions (O

et al. 2007). The induced expression of IDEF1 in

transgenic rice plants would effectively strengthen

the overall regulatory pathway to confer tolerance

of Fe deficiency.

10

exhibited no obvious defects. These differences in the phenotypes of the transformants appear to be related to the distinct nature of the two transcription factors.

5. Future perspectives

In the research described in this paper, we pro- duced various lines of transgenic rice plants with en- hanced tolerance of low Fe availability. Among these lines, three selected lines (gNAS1, gIDS3, and gNAS1- gNAAT) demonstrated tolerance of Fe deficiency in calcareous soil in field trials (Fig. 2, 3). The avail- ability of Fe in rice fields can be severely affected by soil type and redox potential, as well as by numerous other environmental factors. An elaborate combina- tion of previously adopted or new strategies will be needed to produce rice lines with greater tolerance of low Fe availability in problematic soils without a loss of favorable agricultural traits. Manipulation of DMAS genes, which were recently cloned and thus have not been genetically modified, during the biosynthesis of MAs (B

et al. 2006) would be of special inter- est. In addition, further clarification of the underlying mechanisms involved in Fe homeostasis is extremely important, including expressional regulation, secretion of MAs, and metal translocation inside the plants.

Understanding metal homeostasis also paves the way to fortifying rice grains with Fe and Zn. Previous efforts to enhance Fe content in rice grains focused on overproduction of ferritin, a common Fe storage pro- tein in rice grains (G

et al. 1999; Q

et al. 2005;

V

et al. 2003). Our field trials revealed that the gIDS3 line is capable of accumulating more Fe in grains in both calcareous and Andosol paddy fields (S

et al. 2008; M

et al. 2008). Production and characterization of transgenic rice lines with intro- duced biosynthetic genes for MAs and ferritin genes in combination to enhance both Fe uptake and storage is in progress (M

H et al., Ishikawa Prefectural University, unpublished data). Other advanced appli- cations of our knowledge of Fe nutrition include the production of novel antihypertensive substrates. NA, the precursor of MAs, inhibits angiotensin I–convert-

ing enzyme in humans and consequently reduces high blood pressure (K

et al. 1993; S

et al.

1999). We produced a yeast strain that highly accumu- lates NA by introducing the Arabidopsis NAS gene, AtNAS2 (W

et al. 2006). Production and selection of rice lines with elevated levels of NA in the grains by introducing the HvNAS1 gene under the control of a seed-specific promoter of the rice glutelin gene was also achieved (U

et al. 2009).

Public acceptance of genetically modified organ- isms is still low. As a technical way to improve public acceptance, we modified the “marker-free vector” of the Cre/loxP DNA excision system (Z

et al. 2001) to construct a high-capacity binary vector for the trans- formation of rice, from which the sequence sandwiched between two loxP sites (including the selectable mark- er) can be removed by administration of 17β-estradiol ((U

et al. 2009). Many other approaches may in- crease public acceptance of transgenic plants, which have such high potential to increase food production, preserve the environment, and improve human health.

Note added in proof

More recent progress in this area of research has been reviewed by K

T, N

H and N

NK (2010), “Recent insights into iron ho- meostasis and their application in graminaceous crops”, Proceedings of the Japan Academy Series B, Vol. 86, 900-913.

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