The lecturers and the members of the Symposium Committee · Research on Plant Breeding, Indian...

ELUCIDATION OF RESISTANCE MECHANISMS TO ABIOTIC STRESSES AND THE APPLICATION FOR

MOLECULAR BREEDING

Report of Symposiumheld on

July 14-15, 2009

Institute of Radiation BreedingNIAS

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

(48th GF Symposium)

III

NARUMI 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 80th 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 1st 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 48th 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. TSUTSUMI

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

Session ⅠChairperson: T. MASUDA

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

　　……………………………………………………………………………………………………… T. FUJIWARA

Session ⅡChairperson: Y.NAGATO

Genetic Regulation of Lateral Root Development in Arabidopsis

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

Session ⅢChairperson: M. KUSABA

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

　　……………………………………………………………………………………………………………Y. INUKAI

Session ⅣChairperson: T. TANISAKA

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

Session ⅤChairperson: H. HIROCHIKA

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

Session VI

Chairperson: H. NAKAGAWA

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

Session VII

Chairperson: T. ABE

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

Session VIII

Chairperson: Y. SANO

General Discussion

Closing address: Y. NAGATO

PROGRAM

Opening address: H. NAKAGAWA

Congratulatory address: M. ISHIGE

VII

CONTENTS

T. KOBAYASH Genes Involved in Iron Acquisition and Their Application for

H. NAKANISHI Developing New Crops ………………………………………………………………… 1

N. K. NISHIZAWA

T. FUJIWARA Molecular Mechanisms of Boron Transport in Plants and Generation of

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

H. FUKAKI Genetic Regulation of Lateral Root Development in Arabidopsis

– The Role of Auxin Signaling – ………………………………………………………… 27

Y. INUKAI Genetic Improvement of Root System Formation for Adaptation to

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

T. MATSUI Heat-induced Floret Sterility in Rice: Mechanisms of Occurrence and Tolerance. …… 43

N. L. MANIGBAS

E. REDOÑA

X. TIAN

M. YOSHIMOTO

K. SUGIMOTO Genetic Control of Seed Dormancy in Rice …………………………………………… 53

S. MARZOUGI

M. YANO

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

K. YAMAMOTO

I. NARUMI Survival Strategy of a Radioresistant Bacterium: a Review …………………………… 69

General Discussion (in Japanese) ……………………………………………………… 77

1

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

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

Takanori KOBAYASHI1, Hiromi NAKANISHI1 and Naoko K. NISHIZAWA1,2

1 Graduate School of Agricultural and Life Science, The University of Tokyo,

1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan2 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 (MARSCHNER 1995;

MORI 1999). To take up and utilize Fe from the rhizo-

sphere, higher plants have evolved two major strategies

(MARSCHNER 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 (TAK-

AGI 1976), and the resulting Fe(III)–MA complexes are

taken up by roots through a specific transporter in the

plasma membrane (CURIE et al. 2001; TAKAGI 1976).

In calcareous soils, Strategy II is more efficient

than Strategy I (MARSCHNER 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 Takanori KOBAYASHI, Hiromi NAKANISHI and Naoko K. NISHIZAWA

tolerant of Fe deficiency than other graminaceous plants

(MA et al. 1999; MORI and NISHIZAWA 1987; RÖMHELD

and MARSCHNER 1986; TAKAGI 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 (KAWAI et al. 1988; MA and NOMO-

TO 1993; MA et al. 1999; MORI and NISHIZAWA 1987;

SHOJIMA et al. 1990). Methionine is the precursor of

MAs (MORI and NISHIZAWA 1987) and is adenosylated

by S-adenosylmethionine (SAM) synthetase (SAMS).

Nicotianamine synthase (NAS) catalyzes the trimeriza-

tion of SAM into nicotianamine (NA) (HIGUCHI et al.

1994). All higher plants, including non-graminaceous

plants, have a biosynthetic pathway that lets them syn-

thesize NA (LING et al. 1999; NOMA and NOGUCHI

1976), which serves as a common metal chelator in-

volved in the internal transport of various micronutri-

ents, including Fe and zinc (Zn) (HELL and STEPHAN

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.

3GENES INVOLVED IN IRON ACQUISITION AND THEIR APPLICATION FOR DEVELOPING NEW CROPS

2003; TAKAHASHI et al. 2003). NA aminotransferase

(NAAT) catalyzes the first step specific to gramina-

ceous plants: transamination of NA to produce the 3’’-

oxo intermediate (KANAZAWA et al. 1995; SHOJIMA et

al. 1990). DMA synthase (DMAS) subsequently reduc-

es the 3’’-oxo form into DMA (BASHIR 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 (HIGUCHI et al.

1994) and enzyme purification from Fe-deficient barley

roots (HIGUCHI et al. 1999). Two barley NAAT genes,

HvNAAT-A and HvNAAT-B, were also cloned through

the establishment of an enzyme activity assay (OHATA

et al. 1993) and enzyme purification (KANAZAWA et al.

1995; TAKAHASHI et al. 1999). Expression of HvNAS1,

HvNAAT-A, and HvNAAT-B is strongly induced by Fe

deficiency and occurs almost exclusively in the roots

(HIGUCHI et al. 1999; TAKAHASHI 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 (HIGUCHI et

al. 1996; KANAZAWA 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 (NAKANISHI et al. 1993;

OKUMURA 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; NAKANI-

SHI et al. 2000). IDS3 was further confirmed to be MA

synthase by introducing the barley IDS3 gene into rice

(KOBAYASHI 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. SUZUKI et al.

1998). The corresponding genes, HvFDH and HvAPT,

were subsequently cloned (ITAI et al. 2000; SUZUKI 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 (MORI

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 (MA et al. 1995). We also applied a

revised differential-hybridization screening, and identi-

fied iron-deficiency-induced (IDI) genes in barley roots

(YAMAGUCHI et al. 2000a, 2000b, 2002). IDI1 and IDI2

putatively encode enzymes that catalyze steps in the

methionine cycle (KOBAYASHI et al. 2005; M. SUZUKI

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 (NEGISHI et al. 2002; M. SUZUKI 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 (BASHIR et al. 2006).


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 (AKIYAMA

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

(HIEI 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 (HIGUCHI 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 (KOBAYASHI 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

(KOBAYASHI 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 (HIGUCHI et al.

2001; KOBAYASHI et al. 2001; TAKAHASHI 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; TAKAHASHI 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 (TAKAHASHI 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 (TAKAHASHI 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; MORIKAWA 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. SUZUKI

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-


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 (Eh) 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 Eh with time is thought

to have resulted in the absorption of ferrous ions via the

ferrous transporter OsIRT1 (see section 4.1 for details;

ISHIMARU 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: SUZUKI et al. (2008).


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 Eh 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

Eh fell below 0 mV (MORIKAWA 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. EIDE et

al. (1996) isolated the Arabidopsis IRT1 gene, which

is the dominant ferrous Fe transporter in the Fe-up-

take process (VERT 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 (BUGHIO et al. 2002; ISHIMARU 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 Fe2+. Thus, rice plants possess

a system other than Strategy II for Fe uptake, which is

based on the secretion of MAs (ISHIMARU et al. 2006).

In contrast to their ferrous transporting ability, Fe-de-

ficient rice roots do not show increased ferric-chelate

reductase activity (ISHIMARU 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: SUZUKI et al. (2008).


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 Eh decreases (ISHIMARU et al. 2006; SUZUKI

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; OKI 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 (OKI 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 (OKI et

al. 2004).

Another concern in relation to the introduction of

exogenous reductase genes into rice was the choice of

an appropriate promoter. VASCONCELOS 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 (CONNOLLY

et al. 2003; VERT 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 (ISHIMARU 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: ISHIMARU et al. (2007).


controls (Fig. 4b, c; ISHIMARU 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 (BASHIR

et al. 2006; KOBAYASHI and NISHIZAWA 2008; KO-

BAYASHI et al. 2005; MORI 1999; NEGISHI 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 (YOSHIHARA 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; KOBAYASHI 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

(KOBAYASHI 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 (DUCOS et al. 2005; KOBAYASHI 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 (KOBAYASHI et al. 2007; OGO

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; KO-

BAYASHI et al. 2007).

To clarify the molecular mechanisms that regulate

Fe acquisition, we also characterized the Fe-deficiency-


induced transcription factors. Microarray analyses re-

vealed the upregulation of several transcription factor

genes in barley and rice (NEGISHI et al. 2002; OGO 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 (OGO et al. 2006). The core

sequence for OsIRO2 binding was determined to be

CACGTGG (OGO 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 (OGO 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 (OGO et al. 2007). Importantly, OsIRO2

itself possesses multiple IDEF1-binding core sequenc-

es in its promoter region and is positively regulated by

IDEF1 (KOBAYASHI 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; KOBAYASHI et al. 2007; OGO 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: KOBAYASHI et

al. (2007).

Fig. 6. Proposed regulatory network for the induction of

Fe-deficiency-responsive genes via IDEF1, IDEF2,

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

(KOBAYASHI et al. 2007; OGO 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 (OGO

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.


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 (BASHIR 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 (GOTO et al. 1999; QU et al. 2005;

VASCONCELOS 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

(SUZUKI et al. 2008; MASUDA 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 (MASUDA 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 (KINOSHITA et al. 1993; SHIMIZU et al.

1999). We produced a yeast strain that highly accumu-

lates NA by introducing the Arabidopsis NAS gene,

AtNAS2 (WADA 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 (USUDA 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 (ZUO 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

((USUDA 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 KOBAYASHI T, NAKANISHI H and

NISHIZAWA 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.

References

1. AKIYAMA, K., NAKAMURA, S., Suzuki, T., WISNIEWSKA,

I., SASAKI, N. and KAWASAKI, S. 1997. Development of

a system of rice transformation with long genome inserts

for their functional analysis for positional cloning. Plant

and Cell Physiology Supplement 38: s94.

2. BASHIR, K., INOUE, H., NAGASAKA, S., TAKAHASHI, M.,

NAKANISHI, H., MORI, S. and NISHIZAWA, N.K. 2006.

Cloning and characterization of deoxymugineic acid

synthase genes from graminaceous plants. Journal of

Biological Chemistry 43: 32395-32402.

3. BUGHIO, N., YAMAGUCHI, H., NISHIZAWA, N.K., NAKAN-


ISHI, H. and MORI, S. 2002. Cloning an iron-regulated

metal transporter from rice. Journal of Experimental

Botany 53: 1677-1682.

4. CONNOLLY, E.L., CAMPBELL, N.H., GROTZ, N., PRICH-

ARD, C.L. and GUERINOT, M.L. 2003. Overexpression

of the FRO2 ferric chelate reductase confers tolerance

to growth on low iron and uncovers posttranscriptional

control. Plant Physiology 133: 1102-1110.

5. CURIE, C., PANAVIENCE, Z., LOULERGUE, C., DELLAPOR-

TA, S.L., Briat, J.F. and Walker, E.L. 2001. Maize yellow

stripe1 encodes a membrane protein directly involved in

Fe(III) uptake. Nature 409: 346-349.

6. DUCOS, E., FRAYSSE, Å.S. and BOUTRY, M. 2005. Nt-

PDR3, an iron-deficiency inducible ABC transporter in

Nicotiana tabacum. FEBS Letters 579: 6791-6795.

7. EIDE, D., BRODERIUS, M., FETT, J. and GUERINOT, M.L.

1996. A novel iron-regulated metal transporter from

plants identified by functional expression in yeast. Pro-

ceedings of the National Academy of Sciences USA 93:

5624-5628.

8. GOTO, F., YOSHIHARA, T., SHIGEMOTO, N., TOKI, S. and

TAKAIWA, F. 1999. Iron fortificaton of rice seed by the

soybean ferritin gene. Nature Biotechnology 17: 282-

286.

9. HELL, R. and STEPHAN, U.W. 2003. Iron uptake, traffick-

ing and homeostasis in plants. Planta 216: 541-551.

10. HIEI, Y., OHTA, S., KOMARI, T. and KUMASHIRO, T. 1994.

Efficient transformation of rice (Oryza sativa L.) me-

diated by Agrobacterium and sequence analysis of the

boundaries of the T-DNA. The Plant Journal 6: 271-

282.

11. HIGUCHI, K., KANAZAWA, K., NISHIZAWA N.K., CHINO,

M. and MORI, S. 1994 Purification and characterization

of nicotianamine synthase from Fe deficient barley roots.

Plant and Soil 165: 173-179.

12. HIGUCHI, K., KANAZAWA, K., NISHIZAWA, N.K. and

MORI, S. 1996. The role of nicotianamine synthase in

response to Fe nutrition status in Gramineae. Plant and

Soil 178: 171-177.

13. HIGUCHI, K., SUZUKI, K., NAKANISHI, H., YAMAGUCHI,

H., NISHIZAWA, N.K. and Mori, S. 1999. Cloning of

nicotianamine synthase genes, novel genes involved in

the biosynthesis of phytosiderophores. Plant Physiology

119: 471-479.

14. HIGUCHI, K., WATANABE, S., TAKAHASHI, M., KAWASA-

KI, S., NAKANISHI, H., NISHIZAWA, N.K. and MORI, S.

2001. Nicotianamine synthase gene expression differs in

barley and rice under Fe-deficient conditions. The Plant

Journal 25: 159-167.

15. ISHIMARU, Y., KIM, S., TSUKAMOTO, T., OKI, H., KO-

BAYASHI, T., WATANABE, S., MATSUHASHI, S., TAKA-

HASHI, M., NAKANISHI, H., MORI, S. and NISHIZAWA,

N.K. 2007. Mutational reconstructed ferric chelate re-

ductase confers enhanced tolerance in rice to iron de-

ficiency in calcareous soil. Proceedings of the National

Academy of Sciences USA104: 7373-7378.

16. ISHIMARU, Y., SUZUKI, M., TSUKAMOTO, T., SUZUKI, K.,

NAKAZONO, M., KOBAYASHI, T., WADA, Y., WATANABE,

S., MATSUHASHI, S., TAKAHASHI, M., NAKANISHI, H.,

MORI, S. and NISHIZAWA, N.K. 2006. Rice plants take up

iron as an Fe3+-phytosiderophore and as Fe2+. The Plant

Journal 45: 335-346.

17. ITAI, R., SUZUKI, K., YAMAGUCHI, H., NAKANISHI, H.,

NISHIZAWA, N.K., YOSHIMURA, E. and MORI, S. 2000.

Induced activity of adenine phosphoribosyltransferase

(APRT) in Fe-deficient barley roots: a possible role for

phytosiderophore production. Journal of Experimental

Botany 51: 1179-1188.

18. KANAZAWA, K., HIGUCHI, K., NISHIZAWA, N.K., FUSH-

IYA, S., CHINO, M. and MORI, S. 1994. Nicotianamine

aminotransferase activities are correlated to the phyto-

siderophore secretions under Fe-deficient conditions in

Gramineae. Journal of Experimental Botany 45: 1903-

1906.

19. KANAZAWA, K., HIGUCHI, K., NISHIZAWA, N.K., FUSHI-

YA, S. and MORI, S. 1995. Detection of two distinct iso-

zymes of nicotianamine aminotransferase in Fe-deficient

barley roots. Journal of Experimental Botany 46: 1241-

1244.

20. KAWAI, S., TAKAGI, S. and SATO, Y. 1988 Mugineic acid-

family phytosiderophores in root-secretions of barley,

corn and sorghum varieties. Journal of Plant Nutrition

11: 633-642.

21. KINOSHITA, E., YAMAKOSHI, J. and KIKUCHI, M. 1993.

Purification and identification of an angiotensin I-con-

verting enzyme inhibitor from soy sauce. Bioscience,

Biotechnology and Biochemistry 57: 1107-1110.

22. KOBAYASHI, T., NAKANISHI, H., TAKAHASHI, M., KAWA-

SAKI, S., NISHIZAWA, N.K. and MORI, S. 2001. In vivo

evidence that Ids3 from Hordeum vulgare encodes a di-

oxygenase that converts 2’-deoxymugineic acid to mugi-

neic acid in transgenic rice. Planta 212: 864-871.

23. KOBAYASHI, T., NAKAYAMA, Y., ITAI, R.N., NAKANISHI,

H., YOSHIHARA, T., MORI, S. and NISHIZAWA, N.K. 2003.

Identification of novel cis-acting elements, IDE1 and

IDE2, of the barley IDS2 gene promoter conferring iron-

deficiency-inducible, root-specific expression in hetero-

geneous tobacco plants. The Plant Journal 36: 780-793.


24. KOBAYASHI, T., NAKAYAMA, Y., TAKAHASHI, M., IN-

OUE, H., NAKANISHI, H., YOSHIHARA, T., MORI, S. and

NISHIZAWA, N.K. 2004. Construction of artificial pro-

moters highly responsive to iron deficiency. Soil Science

and Plant Nutrition 50: 1167-1175.

25. KOBAYASHI, T. and NISHIZAWA, N.K. 2008. Regulation

of iron and zinc uptake and translocation in rice. In Bio-

technology in agriculture and forestry 62. Rice biology

in the genomics era, ed. HIRANO, H.Y., HIRAI, A., SANO,

Y. and SASAKI, T., 321-335. The Netherlands: Springer.

26. KOBAYASHI, T., OGO, Y., ITAI, R.N., NAKANISHI, H.,

TAKAHASHI, M., MORI, S. and NISHIZAWA, N.K. 2007.

The transcription factor IDEF1 regulates the response to

and tolerance of iron deficiency in plants. Proceedings

of the National Academy of Sciences USA 104: 19150-

19155.

27. KOBAYASHI, T., SUZUKI, M., INOUE, H., ITAI, R.N.,

TAKAHASHI, M., NAKANISHI, H., MORI, S. and NISHIZA-

WA, N.K. 2005. Expression of iron-acquisition-related

genes in iron-deficient rice is co-ordinately induced by

partially conserved iron-deficiency-responsive elements.

Journal of Experimental Botany 56: 1305-1316.

28. LING, H.Q., KOCH, G., BÄUMLEIN, H. and GANAL, M.W.

1999. Map-based cloning of chloronerva, a gene involved

in iron uptake of higher plants encoding nicotianamine

synthase. Proceedings of the National Academy of Sci-

ences USA 96: 7098-7103.

29. MA, J.F. and NOMOTO, K. 1993. Two related biosynthetic

pathways of mugineic acids in Gramineous plants. Plant

Physiology 102: 373-378.

30. MA, J.F., SHINADA, T., MATSUDA, C. and NOMOTO, K.

1995. Biosynthesis of phytosiderophores, mugineic ac-

ids, associated with methionine cycling. Journal of Bio-

logical Chemistry 270: 16549-16554.

31. MA, J.F., TAKETA, S., CHANG, Y.C., IWASHITA, T., MAT-

SUMOTO, H., TAKEDA, K. and NOMOTO, K. 1999. Genes

controlling hydroxylations of phytosiderophores are

located on different chromosomes in barley (Hordeum

vulgare L.). Planta 207: 590-596.

32. MARSCHNER, H. 1995. Mineral Nutrition of Higher

Plants. 2nd edn. London: Academic Press.

33. MARSCHNER, H., RÖMHELD, V. and KISSEL, M. 1986.

Different strategies in higher plants in mobilization and

uptake of iron. Journal of Plant Nutrition 9: 695-713.

34. MASUDA, H., SUZUKI, M., MORIKAWA, K.C., KOBAYASHI,

T., NAKANISHI, H., TAKAHASHI, M., SAIGUSA, M., MORI,

S. and NISHIZAWA, N.K. 2008. Increase in Iron and Zinc

Concentrations in Rice Grains Via the Introduction of

Barley Genes Involved in Phytosiderophore Synthesis.

Rice 1: 100-108.

35. MORI, S. 1999. Iron acquisition by plants. Current Opin-

ion in Plant Biology 2: 250-253.

36. MORI, S. and NISHIZAWA, N. 1987. Methionine as a dom-

inant precursor of phytosiderophores in Graminaceae

plants. Plant and Cell Physiology 28: 1081-1092.

37. MORIKAWA, C.K., SAIGUSA, M., NAKANISHI, H., NISHIZA-

WA, N.K., HASEGAWA, K. and MORI, S. 2004. Co-situs

application of controlled-release fertilizers to alleviate

iron chlorosis of paddy rice grown in calcareous soil.

Soil Science and Plant Nutrition 50: 1013-1021.

38. NAKANISHI, H., OKUMURA, N., UMEHARA, Y., NISHIZA-

WA, N.K., CHINO, M. and MORI, S. 1993. Expression of

a gene specific for iron deficiency (Ids3) in the roots of

Hordeum vulgare. Plant and Cell Physiology 34: 401-

410.

39. NAKANISHI, H., YAMAGUCHI, H., SASAKUMA, T., NISHIZA-

WA, N.K. and MORI, S. 2000. Two dioxygenase genes,

Ids3 and Ids2, from Hordeum vulgare are involved in the

biosynthesis of mugineic acid family phytosiderophores.

Plant Molecular Biology 44: 199-207.

40. NEGISHI, T., NAKANISHI, H., YAZAKI, J., KISHIMOTO,

N., FUJII, F., SHIMBO, K., YAMAMOTO, K., SAKATA, K.,

SASAKI, T., KIKUCHI, S., MORI, S. and NISHIZAWA, N.K.

2002. cDNA microarray analysis of gene expression

during Fe-deficiency stress in barley suggests that polar

transport of vesicles is implicated in phytosiderophore

secretion in Fe-deficient barley roots. The Plant Journal

30: 83-94.

41. NOMA, M. and NOGUCHI, M. 1976. Occurrence of ni-

cotianamine in higher plants. Phytochemistry 15: 1701-

1702.

42. OGO, Y., ITAI, R.N., NAKANISHI, H., INOUE, H., KO-

BAYASHI, T., SUZUKI, M., TAKAHASHI, M., MORI, S. and

NISHIZAWA, N.K. 2006. Isolation and characterization of

IRO2, a novel iron-regulated bHLH transcription factor

in graminaceous plants. Journal of Experimental Botany

57: 2867-2878.

43. OGO, Y., ITAI, R.N., NAKANISHI, H., KOBAYASHI, T.,

TAKAHASHI, M., MORI, S. and NISHIZAWA, N.K. 2007.

The rice bHLH protein OsIRO2 is an essential regulator

of the genes involved in Fe uptake under Fe-deficient

conditions. The Plant Journal 51: 366-377.

44. OGO, Y., KOBAYASHI, T., ITAI, R.N., NAKANISHI, H., KA-

KEI, Y., TAKAHASHI, M., TOKI, S., MORI, S. and NISHIZA-

WA, N.K. 2008. A novel NAC transcription factor IDEF2

that recognizes the iron deficiency-responsive element

2 regulates the genes involved in iron homeostasis in

plants. Journal of Biological Chemistry 283: 13407-


13417.

45. OHATA, T., KANAZAWA, K., MIHASHI, S., NISHIZAWA,

N.K., FUSHIYA, S., NOZOE, S., CHINO, M. and MORI,

S. 1993. Biosynthetic pathway of phytosiderophores in

iron-deficient Graminaceous plants. Development of an

assay system for the detection of nicotianamine amino-

transferase activity. Soil Science and Plant Nutrition 39:

745-749.

46. OKI, H., KIM, S., NAKANISHI, H., TAKAHASHI, M.,

YAMAGUCHI, H., MORI, S. and NISHIZAWA, N.K. 2004.

Directed evolution of yeast ferric reductase to produce

plants with tolerance to iron deficiency in alkaline soils.

Soil Science and Plant Nutrition 50: 1159-1165.

47. OKI, H., YAMAGUCHI, H., NAKANISHI, H. and MORI, S.

1999. Introduction of the reconstructed yeast ferric re-

ductase gene, refre1, into tobacco. Plant and Soil 215:

211-220.

48. OKUMURA, N., NISHIZAWA, N.K., UMEHARA, Y. and

MORI, S. 1991. An iron deficiency-specific cDNA from

barley roots having two homologous cysteine-rich MT

domains. Plant Molecular Biology 17: 531-533.

49. OKUMURA, N., NISHIZAWA, N.K., UMEHARA, Y., OHATA,

T., NAKANISHI, H., YAMAGUCHI, H., CHINO, M. and

MORI, S. 1994. A dioxygenase gene (Ids2) expressed

under iron deficiency conditions in the roots of Hordeum

vulgare. Plant Molecular Biology 25: 705-719.

50. QU, L.Q., YOSHIHARA, T., OOYAMA, A., GOTO, F. and

TAKAIWA, F. 2005. Iron accumulation does not parallel

the high expression level of ferritin in transgenic rice

seeds. Planta 222: 225-233.

51. RÖMHELD, V. and MARSCHNER, H. 1986. Evidence for

specific uptake system for iron phytosiderophores in

roots of grasses. Plant Physiology 80: 175-180.

52. SHIMIZU, E., HAYASHI, A., TAKAHASHI, R., AOYAGI, Y.,

MURAKAMI, T. and KIMOTO, K. 1999. Effects of an-

giotensin I-converting enzyme inhibitor from Ashitaba

(Angelica keiskei) on blood pressure of spontaneously

hypertensive rats. Journal of Nutritional Science and Vi-

taminology 45: 375-383.

53. SHOJIMA, S., NISHIZAWA, N.K., FUSHIYA, S., NOZOE, S.,

IRIFUNE, T. and MORI, S. 1990. Biosynthesis of phyto-

siderophores: in vitro biosynthesis of 2’-deoxymugineic

acid from L-methionine and nicotianamine. Plant Physi-

ology 93: 1497-1503.

54. SUZUKI, K., ITAI, R., SUZUKI, K., NAKANISHI, H.,

NISHIZAWA, N.K,. YOSHIMURA, E. and MORI, S. 1998.

Formate dehydrogenase, an enzyme of anaerobic metab-

olism, is induced by Fe-deficiency in barley roots. Plant

Physiology 116: 725-732.

55. SUZUKI, M., MORIKAWA, K.C., NAKANISHI, H., TAKA-

HASHI, M., SAIGUSA, M., MORI, S. and NISHIZAWA, N.K.

2008. Transgenic rice lines that include barley genes

have increased tolerance to low iron availability in a cal-

careous paddy soil. Soil Science and Plant Nutrition 54:

77-85.

56. SUZUKI, M., TAKAHASHI, M., TSUKAMOTO, T., WATA-

NABE, S., MATSUHASHI, S., YAZAKI, J., KISHIMOTO, N.,

KIKUCHI, S., NAKANISHI, H., MORI, S. and NISHIZAWA,

N.K. 2006. Biosynthesis and secretion of mugineic acid

family phytosiderophores in zinc-deficient barley. The

Plant Journal 48: 85-97.

57. TAKAGI, S. 1976. Naturally occurring iron-chelating

compounds in oat- and rice-root washings. Soil Science


58. TAKAHASHI, M., NAKANISHI, H., KAWASAKI, S., NISHIZA-

WA, N.K. and MORI, S. 2001. Enhanced tolerance of rice

to low iron availability in alkaline soils using barley

nicotianamine aminotransferase genes. Nature Biotech-

nology 19: 466-469.

59. TAKAHASHI, M., TERADA, Y., NAKAI, I., NAKANISHI, H.,

YOSHIMURA, E., MORI, S. and NISHIZAWA, N.K. 2003.

Role of nicotianamine in the intracellular delivery of

metals and plant reproductive development. The Plant

Cell 15: 1263-1280.

60. TAKAHASHI, M., YAMAGUCHI, H., NAKANISHI, H., SHIO-

IRI, T., NISHIZAWA, N.K. and MORI, S. 1999. Cloning two

genes for nicotianamine aminotransferase, a critical en-

zyme in iron acquisition (Strategy II) in graminaceous

plants. Plant Physiology 121: 947-956.

61. USUDA, K., WADA, Y., ISHIMARU, Y., KOBAYASHI, T.,

TAKAHASHI, M., NAKANISHI, H., NAGATO, Y., MORI, S.

and NISHIZAWA, N.K. 2009. Genetically engineered rice

containing larger amounts of nicotianamine to enhance

the antihypertensive effect. Plant Biotechnology Journal

7: 87-95.

62. VASCONCELOS, M., DATTA, K., OLIVA, N., KHALEKUZ-

ZAMAN, M., TORRIZO, L., KRISHNAN, S., OLIVEIRA, M.,

GOTO, F. and DATTA, S.K. 2003. Enhanced iron and zinc

accumulation in transgenic rice with the ferritin gene.

Plant Science 164: 371-378.

63. VASCONCELOS, M., MUSETTI, V., Li, C.M., DATTA, S.K.

and GRUSAK, M.A. 2004. Functional analysis of trans-

genic rice (Oryza Sativa L.) transformed with an Arabi-

dopsis thaliana ferric reductase (AtFRO2). Soil Science


64. VERT, G., BRIAT, J.F. and CURIE, C. 2003. Dual regu-

lation of the Arabidopsis high-affinity root iron uptake

system by local and long-distance signals. Plant Physiol-


ogy 132: 796-804.

65. VERT, G., GROTZ, N., DEDALDECHAMP, F., GAYMARD,

F., GUERINOT, M.L., BRIAT, J.F. and CURIE, C. 2002.

IRT1, an Arabidopsis transporter essential for iron up-

take from the soil and plant growth. The Plant Cell 14:

1223-1233.

66. WADA, Y., KOBAYASHI, T., TAKAHASHI, M., NAKANISHI,

H., MORI, S. and NISHIZAWA, N.K. 2006. Metabolic en-

gineering of Saccharomyces cerevisiae producing nicoti-

anamine: potential for industrial biosynthesis of a novel

antihypertensive substrate. Bioscience, Biotechnology

and Biochemistry 70: 1408-1415.

67. YAMAGUCHI, H., NAKANISHI, H., NISHIZAWA, N.K. and

MORI, S. 2000a. Induction of the IDI1 gene in Fe-defi-

cient barley roots: a gene encoding putative enzyme that

catalyses the methionine salvage pathway for phytosid-

erophore production. Soil Science and Plant Nutrition

46: 1-9.

68. YAMAGUCHI, H., NAKANISHI, H., NISHIZAWA, N.K. and

MORI, S. 2000b. Isolation and characterization of IDI2,

a new Fe-deficiency induced cDNA from barley roots,

which encodes a protein related to the α subunit of eu-

karyotic initiation factor 2B (eIF2B α). Journal of Ex-

perimental Botany 51: 2001-2007.

69. YAMAGUCHI, H., NISHIZAWA, N.K., NAKANISHI, H. and

MORI, S. 2002. IDI7, a new iron-regulated ABC trans-

porter from barley roots, localizes to the tonoplast. Jour-

nal of Experimental Botany 53: 727-235.

70. YOSHIHARA, T., KOBAYASHI, T., GOTO, F., MASUDA,

T., HIGUCHI, K., NAKANISHI, H., NISHIZAWA, N.K. and

MORI, S. 2003. Regulation of the iron-deficiency respon-

sive gene, Ids2, of barley in tobacco. Plant Biotechnol-

ogy 20: 33-41.

71. ZUO, J., NIU, Q.W., MØLLER, S.G. and CHUA, N.H.

2001. Chemical-regulated, site-specific DNA excision in

transgenic plants. Nature Biotechnology 19: 157-161.


質疑応答司会：どうもありがとうございました。せっかくですのでご質問を受け付けたいと思いますが，どなたか。ご質問の際は，後で記録に残るので，ご所属をまずおっしゃってください。深城：神戸大学の深城と申します。前半のムギネ酸のお話なんですけども， ₁ 回キレートして，鉄をキレートして取り込んだムギネ酸というのは，再びリクルートされてまた出ていくってことはあるんでしょうか。西澤：それは可能性としてはあると思います。実は取り込まれた三価鉄ムギネ酸が，その後どうなるかについては全く分かっていません。多分そのままの形で，導管に運ばれて，鉄ムギネ酸の形で地上部に運ばれるっていうことが ₁つあるかと思います。稲の導管液には鉄もムギネ酸類も検出されます。けれども，もしかすると，三価鉄は還元されて二価鉄となり，ニコチアナミンに二価鉄として受け渡されて，深城：受け渡して，はい。西澤：ムギネ酸は外にもう ₁回出るというようなこともあるかもしれません。深城：そうですか。西澤：でもその点については全く分かっていません。深城：ありがとうございました。西澤：また，二価鉄のトランスポーターで吸収した二価鉄の場合は，すぐにニコチアナミンがキャッチして，ニコチアナミン鉄にする。二価鉄はフリーで存在すると，生体にとって危険ですから，ニコチアナミン鉄で安全な形にして，稲の体内を運んでいるのではないかと思います。深城：ありがとうございました。はい。

司会：ほかにございませんでしょうか。草場：広島大学の草場です。 ₃つの鉄耐性の戦略をご紹介いただきましたが， ₂つは，鉄も吸収をアップするというか，そういう戦略で，最後の ₃ つ目の戦略は，やはりその，転写因子をオーバーエクスプレスすることで，その鉄の吸収をあげるようなシステムが働いているということですね。西澤：はい。最後の OsIRO ₂ の過剰発現ですけれども，OsIRO ₂ が制御するのは，かなりの数の鉄欠乏誘導性の遺伝子です。その中の ₁つにOsYSL15もありますから，吸収も活性化してると思いますし，それ以外にもニコチアナミン合成酵素も制御してます。ですから，ニコチアナミンをたくさん作ることによって，ニコチアナミン鉄のような形で鉄の体内移行，そういうものを強化しているということも考えられると思います。草場：ありがとうございました。司会：ほかにございませんでしょうか。岡崎：新潟大学の岡崎と申しますけれども。大変素晴らしいご研究で，大変感動して聞きましたけども。西澤：ありがとうございます。岡崎：実用化のプロセスとか，あるいは特許関係とか，そういうことはどういうふうにやってますか。西澤：はい。最初のムギネ酸類の合成を強化した稲に関しては，ニコチアナミン合成酵素とニコチアナミンアミノ基転移酵素，あるいはムギネ酸合成酵素を強化した稲については，隔離圃場での検定が終わっていますので，今後，本来であればもう少し隔離圃場で検定して，それを一般圃場に出して，というプロセスを踏んで実用

特別講演「植物の鉄栄養制御の分子機構とその応用」

西　澤　直　子

石川県立大学


化に結び付けたいと考えておりますけれども，まだ隔離圃場を終えた段階で，それから先には進んでいません。それから，特許の問題ですけれども，ニコチアナミン合成酵素を含めて，ここに今ご紹介した遺伝子のほとんどについては，科学技術振興機構，CRESTの資金で研究されたもので，科学技術振興機構が遺伝子の特許を取得しております。ですから，日本の特許ということになります。　　国際的にはニコチアナミン合成酵素に関しては，アメリカやオーストラリアのような国でも取得しています。ご承知のように，日本で形質転換イネを実用化するのは，もう非常にハードルが高くて大変で，なんとか日本でやりたいとは思っていますけれども，アメリカにはかなり鉄欠乏の地帯が多いですから，アメリカの会社が大変この特許，この技術に興味を持っているということがあります。実際に接触してきているということです。司会：ほかにございますでしょうか。廣近：生物研の廣近ですけれども， ₂つ質問がありまして，₁つは鉄欠乏のシグナルが，どういった形でその転写因子受け取られるのかということと，もう ₁つは転写因子を使った，いくつかGMが作られてますけれども，その際，生育阻害とかしばしば見られるんですけれども，この場合は特にそういった問題起こってないんでしょうか。西澤：最初の点ですけれども，それはもう私達も

本当に明らかにしたいところで，IDEF ₁ と ₂は，発現量は低いけれども恒常的に存在しますから，どういう形で鉄欠乏のシグナルを受け取って働いているかというのは，今後どうしても明らかにしていきたいと思っています。けれども現段階では分かっていません。それから ₂番目の転写因子の過剰発現ですけれども，最初IDEF ₁ を単純に 35Sのプロモーターで発現させたら，生育阻害が起こりました。ですから，IDS ₂ ，ムギネ酸類水酸化酵素遺伝子ですが，そのプロモーター，鉄欠乏によって誘導がかかるプロモーターを使って形質転換イネを作りましたら，クロロシスになりにくい，鉄欠乏に強い，特に初期生育期に強いという稲ができました。IRO ₂ の場合は，35Sのプロモーターを使って，過剰発現しただけで強くなりました。というのは，IRO ₂ そのものは IDEF ₁ によって制御されていて，鉄欠乏になった時に発現が高まります。35Sで発現させてあらかじめ高い状態にしておけば，強く，早く鉄欠乏にレスポンスして耐性になるということだと思います。IRO

₂ の場合は 35Sのプロモーターでも特に問題はありませんでした。司会：よろしいでしょうか。そろそろ時間となりましたので，これで終わりにしたいと思います。西澤先生，どうも素晴らしいご紹介，

（拍手）西澤：ありがとうございました。司会：ありがとうございました。

17

Introduction

Boron (B) has been recognized as an essential

element for plants for nearly a century (WARINGTON

1923). B deficiency reduces both crop quality and

yields, and has been reported in more than 70 countries

throughout the world (SHORROCKS 1997). In general, B

deficiency affects the newly growing portions of plants.

B deficiency symptoms include the cessation of root

elongation, reduced leaf expansion, and loss of fertil-

ity (LOOMIS and DURST 1992; MARSCHNER 1995; Dell

and HUANG 1997). B deficiency seems to affect cell

elongation rather than cell division (DELL and HUANG

1997). B deficiency also affects membrane function and

metabolic activity (BOLANOS et al. 2004). Conversely,

excess B supply can also reduce crop productivity, as

high levels of B are toxic to living organisms, includ-

ing plants (NABLE et al. 1997). Typical symptoms of B

toxicity include necrosis of leaf margins, resulting from

B accumulation along the transpiration stream. Here, I

review our current understanding of molecular mecha-

nisms of B transport and the use of B transporters for

generation of B-stress–tolerant plants.

Identification of BOR1

It was long believed that B transport in plants is

a passive process, i.e., B is taken up and transported

along concentration gradients. No channel or transport-

er molecule responsible for B transport was identified

until the end of the 20th century.

The first B transporter was identified through the

analysis of an Arabidopsis thaliana mutant, bor1-1.

The bor1-1 mutant was found by Prof. Satoshi Naito

at Hokkaido University and is sensitive to B deficiency

(NOGUCHI et al. 1997). Expansion of the upper leaves

of the mutant was inhibited and the plants failed to

set seeds when plants were grown in the presence of

3 µM boric acid, whereas wild-type plants grew nor-

mally at this B concentration. Leaf expansion of bor1-1

plants recovered when plants were supplemented with 30

µM boric acid, and 150 µM boric acid rescued fertility.

B concentrations in the rosette leaves and the upper por-

tions of the inflorescences were reduced in bor1-1 mutant

plants compared to those of wild-type plants (NOGUCHI

et al. 1997). Reduction of B concentration in bor1-1 was

greater in the shoots than in the roots, indicating that the

bor1-1 mutant is defective in B translocation from roots

to shoots. This is consistent with the observation that the

extent of growth reduction in bor1-1 roots was less than

that in shoots (TAKANO et al. 2001).

To understand the cause of reduced B concen-

tration in leaves of bor1-1, tracer experiments were

conducted. B concentrations in the sap from root cells

increased linearly in proportion to the increase of B

concentration in the medium in both wild-type plants

and the bor1-1 mutant (TAKANO et al. 2002). In con-

trast, B concentrations in the sap from xylem clearly

differed between the two genotypes. A combination

of a linear curve and a saturated curve was present

in B concentrations in xylem sap in wild-type plants,

whereas saturation was not evident in the bor1-1 mu-

tant. These results demonstrated that A. thaliana plants

use a passive process for B uptake into root cells. On

the other hand, an active mechanism to transport B out

of the cell into xylem against concentration gradients is

present in the wild-type plants and missing in the bor1-


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

Toru FUJIWARA

Biotechnology Research Center, The University of Tokyo, Yayoi Bunkyo-ku Tokyo 113-8657, Japan

18 Toru FUJIWARA

1 mutant plants. This is likely to be the cause of de-

creased B translocation from roots to shoots. bor1-1 is

also defective in preferential B translocation into young

leaves (TAKANO et al. 2001). The proportion of B dis-

tribution from the xylem into young leaves was lower in

bor1-1 than in wild-type plants, indicating that BOR1

functions to preferentially transport B into the growing

portions of shoots.

BOR1 was cloned using map-based cloning and

found to be identical to At2g47160. BOR1 has a high

similarity to anion-exchanger proteins in animals.

When BOR1 fused with GFP was transformed into to-

bacco cells by particle bombardment and transiently ex-

pressed, GFP fluorescence was observed at the periph-

ery of the cells, suggesting that BOR1 is localized to

the plasma membrane. BOR1 is capable of transporting

B in yeast (TAKANO et al. 2002). Interestingly BOR1 is

an efflux-type B transporter (TAKANO et al. 2002). Al-

though the chemical form of the transport substrate for

BOR1 has not been experimentally determined, borate

anion is more likely than boric acid, as suggested by

electrophysiology results for NaBC1, a human homo-

log of BOR1 (PARK et al. 2004).

BOR1 is strongly expressed in cells surrounding

the xylem. BOR1 is likely to be involved in effluxing B

from the symplasm to the xylem, resulting in efficient

xylem loading of B (TAKANO et al. 2002).

BOR1 degradation via endocytosis in response to

high B supply

Transcript accumulation of BOR1 was not sig-

nificantly changed under various B conditions in A.

thaliana plants (TAKANO et al. 2005a); however, ac-

cumulation of BOR1 protein decreased under high B

supply in both roots and shoots (TAKANO et al. 2005b).

B translocation from roots to shoots also decreased

upon high B supply (TAKANO et al. 2005a). These re-

sults showed that BOR1 accumulation is regulated at

the posttranscriptional level. This regulation was also

observed in transgenic A. thaliana lines overexpressing

BOR1-GFP under the control of the cauliflower mosaic

virus (CaMV) 35S RNA promoter, a constitutive pro-

moter (TAKANO et al. 2005a). In the transgenic lines,

BOR1-GFP mRNA accumulated constitutively, where-

as BOR1-GFP protein accumulated to high levels only

when B supply was limited.

TAKANO et al. (2005a) demonstrated BOR1 deg-

radation via endocytosis as the first example of directed

plasma membrane protein degradation in plants. In the

roots of transgenic lines expressing BOR1-GFP, GFP

fluorescence was observed at the plasma membrane un-

der low B supply. One hour after transfer to medium

containing high B, GFP fluorescence was observed in

dot-like structures in the plasma membrane; this fluo-

rescence disappeared within two hours. The plasma

membrane proteins GFP-PIP2a and GFP-Lti6b, which

are not involved in B transport, remained in the plasma

membrane at a constant level irrespective of B condi-

tions, suggesting that the degradation mechanism that

operates under high B supply is specific to BOR1.

Furthermore, the BOR1-GFP found in the dot-like

structures was shown to be co-localized with endocyto-

sis markers FM4-64 and mRFP-Ara7. Treatment with

concanamycin A, a specific inhibitor of V-ATPase,

inhibited BOR1-GFP degradation in vacuoles. After

treatment with Brefeldin A (BFA), which inhibits exo-

cytosis but not endocytosis, BOR1-GFP was observed

in vesicles called BFA compartments under both low-

and high-B conditions. These observations indicate that

BOR1-GFP cycles between the plasma membrane and

early endosome irrespective of B conditions. In the

presence of high B supply, BOR1 is trafficked to late

endosome compartments, followed by degradation in

vacuoles. The biological significance of BOR1 degra-

dation in the presence of high B levels is likely to be

prevention of B overaccumulation in the aerial portion

of the plant.

A. thaliana NIP5;1, a channel for boric acid uptake

under B-limited conditions

As described above, physiological experiments

have demonstrated the presence of a channel-mediated

B transport system in plants. TAKANO et al. (2006) iden-

tified NIP5;1, a member of the major intrinsic proteins

(MIPs), as a boric acid channel for efficient B uptake in

A. thaliana roots and showed its physiological impact

19BORON TRANSPORT IN PLANTS

for plant growth under low-B conditions.

MIPs, also known as aquaporins, are a membrane

protein superfamily with six transmembrane regions.

MIPs transport non-charged small molecules such as

glycerol, urea, and ammonia in addition to water (TYER-

MAN et al. 2002). NIP (Nod 26-like Intrinsic Protein) is

one of the four subfamilies of MIPs in plants. Soybean

NOD26 is the founder of this subfamily. NOD26 is lo-

calized to the symbiosome membrane and is capable

of transporting water, glycerol, and ammonia (WAL-

LACE et al. 2006). It has been suggested that NOD26

transports ammonia from rhizobium to plants. On the

other hand, the subcellular localization and physiologi-

cal roles of NIP in non-symbiotic plants were largely

unknown until several years ago.

NIP5;1, was identified through microarray analy-

sis as a gene induced by low-B conditions (TAKANO

et al. 2006). When RNAs isolated from wild-type A.

thaliana roots were treated with normal or low B for

3 days, NIP5;1 transcript accumulation was found to

be highly induced by low B supply. RT-PCR analysis

revealed that NIP5;1 mRNA accumulation in roots in-

creased to more than 10-fold the initial level at 24 h

after the initiation of low B treatment. Treatment with

high B restored the NIP5;1 mRNA level back to normal

within 24 h. In transgenic A. thaliana lines carrying a

NIP5;1 promoter–GUS construct, GUS staining was

strongly observed in the root under low-B conditions.

Under high B conditions, GUS staining was weak, sug-

gesting that NIP5;1 accumulation is regulated by the

promoter region used in the experiment.

The GFP-NIP5;1 fusion protein was localized to

plasma membrane when GFP-NIP5;1 was transiently

expressed in A. thaliana protoplasts. When NIP5;1 was

expressed in Xenopus oocytes for functional analysis,

uptake of boric acid into cells was increased compared

to uptake of water, suggesting that NIP5;1 is a channel

for boric acid (TAKANO et al. 2006).

The function of NIP5;1 in plants was investigated

with T-DNA insertion lines (TAKANO et al. 2006). Two

independent lines in which NIP5;1 had a T-DNA in-

sertion showed severe reduction both in shoot growth

and in root cell elongation under limited B; both shoots

and roots grew normally under normal B levels. The

amounts of B uptake into roots were increased in wild-

type plants under low B supply compared to those under

high B supply, whereas this increase of B uptake was

not observed in the nip5;1-1 T-DNA insertion mutant.

These observations demonstrated that NIP5;1 is essen-

tial for B uptake into root cells to support normal plant

growth under B limitation (TAKANO et al. 2006). Given

its similarity to aquaporins, NIP5;1 is likely to transport

boric acid along a concentration gradient, contributing

to the B requirements of shoot and root growth.

Nine NIP members in the A. thaliana genome are

classified into two subgroups, each of which may have

a different specificity of transport substrates. Rice Lsi1,

also called OsNIP4;1, is a member of the NIP subgroup

containing NIPs present in rice and maize. Lsi1 is re-

quired for the uptake of silicic acid (Si(OH)4) in roots

(MA et al. 2006). Plants are likely to use NIPs for trans-

port of non-charged small nutrient molecules such as

boric acid, ammonia, and silicic acid.

Combination of BOR1 and NIP5;1 for efficient B

transport under B limitation

Through molecular genetic studies of A. thaliana,

BOR1 and NIP5;1 were identified as an efflux-type B

transporter and a channel for boric acid, respectively.

Both are required for normal plant growth under a lim-

ited supply of B. These two types of transporters have

different functions in boric acid transport. Under B

limitation, BOR1 in the pericycle cells exports borate

into the xylem against concentration gradients. NIP5;1

stimulates influx of boric acid from the external me-

dium into root cells; this is possible because NIP5;1 is

expressed in the epidermis and cortex, which are out-

side of the Casparian strips. BOR1 is likely to gener-

ate a concentration gradient between the root cells and

the medium, which is necessary for B influx into root

cells mediated by the channel molecule NIP5;1. If this

hypothesis is correct, then BOR1 function would be

required for efficient transport of boric acid through

NIP5;1, because NIP5;1 is a passive channel for boric

acid and a B concentration gradient would be required

as a driving force.

20 Toru FUJIWARA

It is intriguing that NIP5;1 expression is regulated

at the level of mRNA accumulation, whereas BOR1 ac-

cumulation is regulated through protein degradation. It

is not clear why plants contain two different systems

to regulate flow of B or how beneficial these different

regulation systems are for the plant. Moreover, it is hard

to imagine a situation in which plants would be exposed

abruptly to a high concentration of B. But considering

that BOR1 is the transporter that generates concentra-

tion gradients of B, it might be beneficial for plants to

have a mechanism to rapidly downregulate BOR1, to

rapidly shut off the B flow across the cell if necessary.

NIP6;1, a channel for boric acid distribution to

leaves under B limitation

As previously discussed, B in soil is taken up by

roots through NIP5;1, a boric acid channel, and loaded

into the xylem by BOR1, a borate exporter. The func-

tion of NIP6;1, the most similar protein to NIP5;1, was

studied by using a reverse genetics approach (TANAKA

et al. 2008). NIP6;1 facilitates the rapid permeation of

boric acid across the membrane, but it is completely

impermeable to water. NIP6;1 transcript accumula-

tion is elevated in response to B deprivation in shoots,

but not in roots. In transgenic plants, a NIP6;1 pro-

moter–GUS construct was predominantly expressed in

nodal regions of shoots, especially the phloem region

of vascular tissues. Three independent lines containing

T-DNA insertions in the NIP6;1 gene exhibited reduced

expansion of young rosette leaves only under low B

conditions; expansion under high B conditions was nor-

mal. In these mutants, B concentrations decreased in

young rosette leaves but not in old leaves. These results

strongly suggest that NIP6;1 is a boric acid channel

required for proper distribution of boric acid, particu-

larly to young developing shoot tissues. It appears that

NIP6;1 is involved in xylem–phloem transfer of boric

acid at the nodal regions, and that the watertight prop-

erty of NIP6;1 is important for this function (TANAKA

et al. 2008). It is interesting to note that during evolu-

tion, NIP5;1 and NIP6;1 diverged both in terms of the

cell-type specificity of their expression in plant tissues

and their water permeation properties, while maintain-

ing their abilities to be induced under low B and their

boric acid transport activities.

Improvement of plant growth through BOR and

NIP transporters: Development of low-B-tolerant

plants

Polyols including sorbitol have been shown to

enhance B translocation. TAO et al. (1995) introduced

an apple cDNA encoding NADP-dependent sorbitol-6-

phosphate dehydrogenase (S6PDH), a key enzyme for

sorbitol synthesis, into tobacco, which is not normally a

sorbitol producer. S6PDH activity and sorbitol synthe-

sis were confirmed in the transgenic tobacco expressing

S6PDH under the control of the CaMV 35S promoter.

BROWN et al. (1999) reported that a sorbitol-producing

transgenic tobacco line (S11) became tolerant to B de-

ficiency. S11 showed improvement in plant growth and

seed yields under low B conditions, especially when B

was foliar-applied to mature leaves, compared with the

wild-type tobacco (SR1) and a transgenic line carrying

an antisense construct of S6PDH (A4). The transgenic

S11 line did not exhibit visible B-deficiency symptoms,

whereas wild-type (SR1) showed flower bud abortion

and chlorosis of young leaves under foliar application.

Foliar-applied 10B translocation into the plant apex and

seeds was remarkably enhanced in the S11 transgenic

line, suggesting that enhanced B translocation resulted

in improved plant growth and seed yields.

BELLALOUI et al. (1999) also reported enhanced

B uptake in the sorbitol-producing transgenic tobacco

line S11. The total amounts of B uptake and B distri-

bution in meristematic tissues were increased in S11

compared with the wild-type (SR1) and the antisense

transgenic line (A4). Interestingly, sorbitol concentra-

tions and contents in S11 were increased as B concen-

tration in the medium was increased. Enhancement of

sorbitol production results not only in increase of B

translocation from mature leaves to the sink, but also

in increased B transport and tolerance to B deficiency.

However, engineering of sugar composition can affect

basic plant growth properties. For example, transgenic

tobacco engineered to hyperaccumulate sorbitol showed

necrosis and sterility, possibly due to inhibition of ino-


sitol biosynthesis (SHEVELEVA et al. 1998). DEGUCHI et

al. (2004) reported that introduction of an S6PDH con-

struct into Japanese persimmon conferred salt tolerance

but also dwarfism. Transgenic rice carrying S6PDH

showed increased sorbitol production and B transloca-

tion (BELLALOUI et al. 2003); however, wild-type rice

and the control transgenic plants also produced sorbi-

tol, and improvement of plant growth in the sorbitol-ac-

cumulating transgenic rice was not evident. Producing

a sorbitol is one strategy to confer B tolerance to B de-

ficiency-sensitive plants; however, it is becoming clear

that sorbitol production affects other sugar metabolism

or can even make plants sensitive to B toxicity. There-

fore, it may not be easy to apply this method to various

types of crops for plant improvement.

Another trial for generation of plants tolerant to B de-

ficiency was intended to enhance primary translocation of

B from roots. MIWA et al. (2006) reported the growth prop-

erties of transgenic A. thaliana lines expressing BOR1 un-

der the control of the CaMV 35S promoter. When BOR1

was overexpressed in A. thaliana plants, improvement of

shoot growth and fertility was observed in the transgenic

lines grown under limited B; this improvement was caused

by increased B translocation to shoots and shoot apex. It

is likely that the xylem loading of B that is normally per-

formed by endogenous BOR1 was enhanced in the trans-

genic lines. The advantage of this approach is that there

is no detrimental effect on plant growth under normal or

even toxic levels of B, probably owing to the degradation

of BOR1 under high-B conditions. This is the first report

of plants that show improved seed yields under nutrient-

deficient conditions as a result of enhanced expression

of an essential mineral nutrient transporter. Since BOR1

homologs are present in a wide variety of plant species

(TAKANO et al. 2002), this transgenic strategy might be

useful in other crops; in addition, BOR1 homologs might

be useful as genetic markers for breeding.

Improvement of plant growth properties through

BOR and NIP transporters: High-B-tolerant plants

produced by BOR4 overexpression

B toxicity is a worldwide problem that impedes

food production in semiarid regions. In south Australia,

more than 10% wheat yield loss was estimated to be

caused by B toxicity. It was reported that overexpression

of BOR4, an A. thaliana paralog of BOR1, is capable of

conferring extreme tolerance to high B toxicity (MIWA

et al. 2007). Three homozygous Arabidopsis transgenic

lines overexpressing AtBOR4 showed remarkable im-

provement of root and shoot growth in the presence of

10 mM B, a concentration lethal to wild-type plants. B

concentrations in roots and shoots were decreased in

these transgenic plant lines in the presence of 3 mM bo-

ric acid. There was no significant difference in growth

between wild-type and transgenic lines under a normal

supply of B. Overexpression of a construct encoding a

BOR4-GFP fusion protein reduced the B concentration

in cells, which was beneficial to maintaining growth

under high-B conditions.

In the transgenic lines expressing a ProBOR4-

BOR4-GFP construct, GFP fluorescence was strongly

detected in the plasma membrane of the distal side of

the root epidermal cells in the elongation zone. Distal

localization of BOR4 is likely to be important for di-

rectional B export from roots to soil to prevent B accu-

mulation in xylem and growing cells. Overexpression

of BOR4 promoted effective exclusion of B out of the

cells, which likely resulted in improvement of growth.

BOT1, a barley homolog of BOR4, may be involved in

high B tolerance (SUTTON et al. 2007), supporting the

importance of this type of B extruder in B tolerance.

Enhancement of B efflux from the roots of crop

plants is expected to result in improvement of crop pro-

ductivity on B-toxic soils found in a number of regions

around the world.

Growth improvement under low-B conditions by

enhanced expression of NIP5;1

As described above, overexpression of the B trans-

porter BOR1 improves shoot growth under low B, but

root growth remains similar to that of wild-type plants.

Moderate enhancement of expression of NIP5;1, but

not overexpression, resulted in improved root elonga-

tion under low-B conditions in A. thaliana. An NIP5;1

activation tag line, which had a T-DNA insertion with

enhancer sequences near the NIP5;1 gene, exhibited

22 Toru FUJIWARA

improved root elongation under the low-B condition,

while overexpression of NIP5;1 by the CaMV 35S RNA

promoter resulted in reduced overall growth. When

moderately enhanced expression of NIP5;1 was com-

bined with overexpression of BOR1, plants with even

greater tolerance to low B were generated (KATO et al.

2009). Furthermore, one of transgenic lines containing

both genes exhibited improved fertility and short-term

B uptake. This represents the first successful improve-

ment of tolerance to B deficiency through modification

of transporters and also reveals the potential of enhanc-

ing expression of a mineral nutrient channel gene to im-

prove growth under nutrient-limited conditions.

Conclusions and perspectives

The studies described in this report have revealed

that NIP5;1 and NIP6;1 play important but distinct

roles in B uptake and distribution, respectively, within

the plant body. Recent success in generating plants tol-

erant to B deficiency by manipulation of NIP5;1 ex-

pression illustrated that NIPs represent targets for plant

breeding for enhancement of growth. Future studies are

expected to broaden and deepen our understanding of

NIPs, which will lead to better technology to develop

and grow plants that are beneficial for humans.

References

1. Bellaloui N., Brown P.H., and Dandekar A.M.

(1999). Manipulation of in vivo sorbitol production al-

ters boron uptake and transport in tobacco. Plant Physi-

ol. 119: 735-742.

2. Bellaloui N., Yadavc R.C., Chern M.S., Hu H., Gil-

len A.M., Greve C., Dandekar A.M., Ronald P.C.,

and Brown P.H. (2003). Transgenically enhanced sorbi-

tol synthesis facilitates phloem-boron mobility in rice.

Physiol. Plant. 117: 79-84.

3. Bolanos L., Lukaszewski K., Bonilla I., and

Blevins D. Why boron? Plant Physiol Biochem 2004;

42: 907-12.

4. Brown P.H., Bellaloui N., Hu H., and Dandekar A.

Transgenically enhanced sorbitol synthesis facilitates

phloem boron transport and increases tolerance of to-

bacco to boron deficiency. Plant Physiol 1999; 119: 17-

20.

5. Deguchi M., Koshita Y., Gao M., Tao R., Tetsumura

T., Yamaki S., and Kanayama Y. Engineered sorbitol

accumulation induces dwarfism in Japanese persimmon.

J Plant Physiol 2004; 161, 1177-84.

6. Dell B., and Huang L.B. Physiological response of

plants to low boron. Plant Soil 1997; 193: 103-20.

7. Kato Y., Miwa K., Takano J., Wada M., and Fujiwara

T. Highly boron deficiency-tolerant plants generated by

enhanced expression of NIP5;1, a boric acid channel.

Plant Cell Physiol 2009; 50: 58-66.

8. Loomis W.D., and Durst R.W. Chemistry and biology

of boron. Biofactors 1992; 3: 229-39.

9. Ma J.F., Tamai K., Yamaji N., Mitani N., Konishi S.,

Katsuhara M., Ishiguro M., Murata Y., and Yano

M. A silicon transporter in rice. Nature 2006; 440: 688-

91.

10. Marschner H. Mineral Nutrition of Higher Plants 2nd

ed. 1995; Academic Press, San Diego, CA.

11. Miwa K., Takano J., and Fujiwara T. Improvement

of seed yields under boron-limiting conditions through

overexpression of BOR1, a boron transporter for xylem

loading, in Arabidopsis thaliana. Plant J 2006; 46: 1084-

91.

12. Miwa K., Takano J., Omori H., Seki M., Shinozaki

K., and Fujiwara T. Plants tolerant of high boron levels.

Science 2007; 318: 1417.

13. Nable R.O., Banelos G.S., and Paull J.G. Boron tox-

icity. Plant Soil 1997; 193: 181-98.

14. Noguchi K., Yasumori M., Imai T., Naito S., Mat-

sunaga T., Oda H., Hayashi H., Chino M., and Fu-

jiwara T. bor1-1, an Arabidopsis thaliana mutant that

requires a high level of boron. Plant Physiol 1997; 115:

901-6.

15. Park M., Li Q., Shcheynikov N., Zeng W., and Mual-

lem S. NaBC1 is a ubiquitous electrogenic Na+-coupled

borate transporter essential for cellular boron homeosta-

sis and cell growth and proliferation. Mol. Cell 2004; 16:

331-341

16. Sheveleva E.V., Marquez S., Chmara W., Zegeer

A., Jensen R.G., and Bohnert H.J. Sorbitol-6-phos-

phate dehydrogenase expression in transgenic tobacco.

High amounts of sorbitol lead to necrotic lesions. Plant

Physiol 1998; 117, 831-9.

17. Shorrocks V.M. The occurrence and correction of bo-

ron deficiency. Plant Soil 1997;193: 121-48.

18. Sutton T., Baumann U., Hayes J., Collins N.C., Shi

B.J., Schnurbusch T., Hay A., Mayo G., Pallotta

M., Tester M., and Langridge P. Boron-toxicity toler-


ance in barley arising from efflux transporter amplifica-

tion. Science 2007; 318: 1446-9.

19. Takano J., Yamagami M., Noguchi K., Hayashi H.,

and Fujiwara T. Preferential translocation of boron to

young leaves in Arabidopsis thaliana regulated by the

BOR1 gene. Soil Sci. Plant Nutr 2001; 47: 345-57.

20. Takano J., Noguchi K., Yasumori M., Kobayashi M.,

Gajdos Z., Miwa K., Hayashi H., Yoneyama T., and

Fujiwara T. Arabidopsis boron transporter for xylem

loading. Nature 2002; 420: 337-40.

21. Takano J., Miwa K., Yuan L., von Wiren N., and

Fujiwara T. Endocytosis and degradation of BOR1, a

boron transporter of Arabidopsis thaliana, regulated by

boron availability. Proc Natl Acad Sci USA 2005a; 102:

12276-81.

22. Takano J., Miwa K., von Wiren N., and Fujiwara T.

Regulation of the boron transporter AtBOR1 by boron

availability. In Plant nutrition for food security, human

health and environmental protection, (Li, C.J. et al., eds).

2005b; Beijing, China: Tsinghua University Press, pp.

138-9.

23. Takano J., Wada M., Ludewig U., Schaaf G., von

Wiren N., and Fujiwara T. The Arabidopsis major in-

trinsic protein NIP5;1 is essential for efficient boron up-

take and plant development under boron limitation. Plant

Cell 2006; 18: 1498-509.

24. Tanaka M., Wallace I.S., Takano J., Roberts D.M.,

and Fujiwara T. NIP6;1 is a boric acid channel for pref-

erential transport of boron to growing shoot tissues in

Arabidopsis. Plant Cell 2008; 20: 2860-75.

25. Tao R., Uratsu S.L., and Dandekar A.M. Sorbitol syn-

thesis in transgenic tobacco with apple cDNA encoding

NADP-dependent sorbitol-6-phosphate dehydrogenase.

Plant Cell Physiol 1995; 36: 525-32.

26. Tyerman S.D., Niemietz C.M., and Bramley H.

Plant aquaporins: multifunctional water and solute

channels with expanding roles. Plant Cell Environ 2002;

25: 173-94.

27. Wallace I.S., Choi W.G., and Roberts D.M. The

structure, function and regulation of the nodulin 26-like

intrinsic protein family of plant aquaglyceroporins. Bio-

chim Biophys Acta 2006; 1758: 1165-75.

28. Warington K. The effect of boric acid and borax on the

broad bean and certain other plants. Ann Bot 1923; 37:

629-72.

24 Toru FUJIWARA

ホウ素輸送の分子機構の解明とホウ素ストレス耐性植物の作出

藤　原　　　徹

東京大学

　ホウ素が植物の必須元素であることが見いだされたのは₈₀年以上前であるが，今世紀に入るまでその生理機能や輸送の分子メカニズムは知られていなかった。シロイヌナズナの変異株を用いた解析によって，ホウ素が細胞壁ペクチンのラムノガラクツロナン II側鎖を架橋することが生理作用の一つであることが明らかにされた。また，ホウ

素を輸送する蛋白質が複数発見され，それらの活性や生理的な役割などが明らかにされ，さらには，これらの輸送体を用いて，ホウ素欠乏や過剰に耐性を示す植物が作出されるようになっている。本稿はこれら，ホウ素の生理作用や輸送についての最近の知見をまとめたものである。


質疑応答司会：どうもありがとうございました。ミュータントのですね，偶然な発見から始まったということですけども，その後，非常にメカニズム解明まで進められたと思います。非常にきれいなご発表だと思います。皆さん，ほか何かご質問等ございましたら，ちょっとお願いしたいと思いますが，いかがでしょうか。はい。マイクの方，ちょっとお願いします。深城：神戸大学の深城です。藤原：どうも。深城：BOR ₁ と BOR ４で，それぞれ役割分担をしているっていうことと，BOR ₁ の方がタンパク質は不安定なもので，で，BOR ４の方はそうではないということですよね。藤原：はい。深城：で，まず BOR ４の方なのですけど，根の表皮細胞の，特にその外側に極性を持って局在化してたってのが，非常に興味深かったのですけども，多分その，恐らくエンドサイトーシスとか，そういったことが係わるのではないかなと思いますが，その極性を持った局在に何か関与するような因子は，何かあるのでしょうか。藤原：ええ，はい。 ₁番と４番は逆側にあるのですね。それで，比較的似たタンパク質です。相同性は結構高くて。で，そうするとまあ，アミノ酸を比較すると，どのアミノ酸が関与しているのかを検討したくなります。で今やっている実験としましては， ₁番と４番を，スワップして，どこをこう変えると内側に行くとか外側に行くっていう実験を，うまくやろうとしておりますが，起こることは ₁番の内のどっかを４番にすると，ちょっと，ちょっと大ざっぱに言い過ぎかもしれませんけども。どっかを４番にすると，分解しにくくなります。で，局在はうまくできません。内側にも外側にも，きれいにならなくなります。そこで，先ほど， ₁ 番の 590

番のリジンっていうお話しました。深城：はい。ありましたね。はい。藤原：あれはあの，局在は変わらずに分解だけ制御されるっていうものです。それ以外のアミノ酸を壊すと，局在も変わり，分解も変わったりします。そのではどうして結局４番は外側にあ

り， ₁番を内側にあるのかっていう疑問は，本当には答えられないのですが，いくつかの阻害剤の実験とかをしますと，恐らく ₁番はですね，これもちょっと不思議なのですが，常に作られて壊されています。ホウ素がたくさんあるときに，エンドサイトーシスを止めるような役割をすると，見えなかった GFPが液胞にはまったりとかっていうことが見えます。だから，常に作って壊している。深城：そうですよね。なるほど。藤原：無駄なことをしている。深城：リサイクリングをずっとやっているのですね。藤原：はい。で，４番の方は，あまりエンドサイトーシスを受けているように見えません。それはひょっとすると，外側にはですね，なんかこう，どういったらいいでしょうか。裏打ちタンパク質というか，なんかとめておくタンパク質があるのかなと思っていますが。じゃあ何がとめているんでしょうっていうのが，多分次の知るべき課題だと思っています。深城：はあ。BOR ４の方は，じゃ，さっきのそのK590に相当するようなところっていうのは，相同性は特にないんですかね。藤原：そこのアミノ酸はリジンではありません。深城：あ，そうですか。藤原：はい。で，そのリジンはユビキチン化されているかもしれないと思っていて，まだその590番が，本当にユビキチン化がされているか，ちょっと調べないといけないのですけども，ま，BOR ₁ がユビキチン化を受けることは分かっています。深城：はい。ありがとうございました。司会：はい。時間の関係もございますが，もうお₁人方ぐらいございましたら，ちょっとご質問受けたいと思いますが，いかがでしょうか。と，よろしいでしょうか。わたしの方からちょっと₁つ，最後のスライドのころだと思いますけれども，BORの４ですね，入れた系統がいくつかありましたよね。藤原：はい。司会：根が非常に， ₁番右の写真は長く伸びていると。

26 Toru FUJIWARA

藤原：はい。司会：あの個体も，上層部の方，地上部も非常にきれいに伸びているわけでしょうか。藤原：はい。そうです。はい。司会：そうしますと，過剰発現することによって，根の方も地上部も両方とも，生育が良分のものがとられるということですね。藤原：そうですね。根の吸収がおかしくなる，根であまり吸収しなくなるというふうに言いましょうかね。吸収したもの吐きだすので。地上

部にホウ素があまり送れなくなって，根も地上部もよく伸びるという，よく育つということだと思います。司会：はい。分かりました。ありがとうございます。それでは時間ですので，藤原先生のですね，ご講演，これで終わりと思います。どうもありがとうございました。藤原：どうもありがとうございました。（拍手）

27

Introduction

In flowering plants, the root system enables the

plant to absorb water and nutrients efficiently and to

sustain the aerial shoots. This branching root system is

developed through the production of many lateral and

adventitious roots. The pattern of the root system dif-

fers depending on the species, and it often changes in

response to environmental stresses (reviewed in MALA-

MY 2005). It is well known that lateral root (LR) forma-

tion is dependent on auxin, but the molecular mecha-

nisms that regulate auxin-mediated LR formation are

still largely unknown. Recent molecular genetic studies

using Arabidopsis thaliana have revealed that the Aux/

IAA (Auxin/Indole-3-Acetic Acid) and ARF (AUXIN

RESPONSE FACTOR) families of transcriptional reg-

ulators are important for auxin-mediated LR formation

(reviewed in FUKAKI et al. 2007; FUKAKI and TASAKA

2009). This article focuses on auxin signaling in the

regulation of LR formation in Arabidopsis.

1. Auxin-mediated lateral root formation in Arabi-

dopsis

As in most eudicot plants, Arabidopsis LRs are

initiated from asymmetric, anticlinal cell divisions in

the root pericycle adjacent to the two protoxylem poles

(protoxylem pericycle) (BARLOW et al. 2004). These

divided cells then undergo periclinal cell divisions to

make a young LR primordium. After a series of cell di-

visions and cell differentiation in the primordium, each

LR primordium develops a root apical meristem, which

promotes subsequent LR growth (MALAMY and BEN-

FEY 1997; CASIMIRO et al. 2003).

In Arabidopsis, application of exogenous auxin

increases the number of LRs, whereas treatment with

auxin transport inhibitors decreases the number of LRs,

indicating that LR formation is dependent on auxin

(LASKOWSKI et al. 1995; REED et al. 1998; CASIMIRO

et al. 2001). In addition, recent molecular genetic stud-

ies using mutants in Arabidopsis and other plants have

shown that LR initiation and subsequent LR primor-

dium development depend on auxin because mutations

affecting auxin biosynthesis, transport, and signaling

have been shown to affect the number of LRs (reviewed

in FUKAKI et al. 2007; FUKAKI and TASAKA 2009).

2. The solitary-root mutant and arf7 arf19 double

mutant in Arabidopsis exhibit severely impaired

LR formation

Among studies of Arabidopsis mutants that exhibit

defective LR formation, studies of the solitary-root (slr)

mutant and the arf7 arf19 double mutant have revealed

that transcriptional regulation by Aux/IAA (Auxin/In-

dole-3-Acetic Acid) proteins, which are repressors of

auxin-responsive transcription, and by ARF (AUXIN

RESPONSE FACTOR) protein is important for LR for-

mation (Figure 1; FUKAKI et al. 2002; OKUSHIMA et al.

2005; WILMOTH et al. 2005).

In general, auxin-responsive gene expression is

regulated by both ARF and Aux/IAA proteins. ARFs

bind to the auxin-responsive element (AuxRE) in the

promoters of the auxin-responsive genes and either ac-

tivate or repress transcription (HAGEN and GUILFOYLE

2002). Among the mutants in the 23 ARF family mem-


Genetic Regulation of Lateral Root Development in Arabidopsis– The Role of Auxin Signaling –

Hidehiro FUKAKI

Department of Biology, Graduate School of Science, Kobe University, Japan

28 Hidehiro FUKAKI

bers in Arabidopsis, only the arf7 arf19 double mutant

exhibits severely impaired LR formation, indicating

that ARF7 and ARF19 positively regulate LR formation

via activation of their downstream genes (OKUSHIMA

et al. 2005; WILMOTH et al. 2005). On the other hand,

Aux/IAAs inactivate ARF function through their inter-

action with the ARFs, thereby repressing auxin-respon-

sive transcription. Aux/IAAs are unstable proteins and

are rapidly degraded by auxin signaling.

In Arabidopsis, auxin signals are captured by the

auxin receptor F-box proteins TRANSPORT INHIBI-

TOR RESPONSE1 (TIR1) and AUXIN SIGNALING

F-BOX (AFB)1, -2, and -3 (DHARMASIRI et al. 2005; KE-

PINSKI and LEYSER 2005). TIR1/AFBs interact with the

Aux/IAA proteins and promote the ubiquitination of the

Aux/IAAs through Skp1-Cullin-F-box (SCF)TIR1/AFB1/2/3

E3 ubiquitin ligase complexes. After ubiquitination, the

Aux/IAAs are degraded by the 26S proteasome. This al-

lows auxin-responsive transcription regulated by ARF,

which can act as either a transcriptional activator or a

transcriptional repressor (reviewed by MOCKAITIS and

ESTELLE 2008). Gain-of-function mutations in Aux/

IAA protein domain II, which is important for Aux/IAA

interactions with SCFTIR1/AFB1/2/3 complexes, block inter-

actions between Aux/IAAs and the SCFTIR1/AFBs com-

plexes, thus increasing the stability of Aux/IAAs and

resulting in constitutive inactivation of ARF functions.

Such gain-of-function mutations in domain II have

been identified in several Aux/IAA genes. Among the

gain-of-function iaa mutants found in the 29 Aux/IAA

family members in Arabidopsis, the axr5/iaa1, shy2/

iaa3, slr/iaa14, crane/iaa18, msg2/iaa19, and iaa28-1

mutants are impaired in LR formation (UEHARA et al.

2008; reviewed in FUKAKI et al. 2007; FUKAKI and TA-

SAKA 2009). In particular, the slr-1/iaa14 mutant has

no LRs due to the inhibition of anticlinal cell divisions

for LR initiation in the protoxylem pericycle (FUKAKI

et al. 2002; VANNESTE et al. 2005), and SLR/IAA14

interacts with ARF7 and ARF19 in the yeast two-hy-

brid assay (FUKAKI et al. 2005), suggesting that the sta-

bilized slr-1/iaa14 protein in the slr-1 mutant may re-

press ARF7/ARF19 functions by interacting with these

ARFs. These studies have shown that LR formation is

regulated by auxin-dependent transcriptional regulation

through ARF7, ARF19, and several Aux/IAAs, includ-

ing SLR/IAA14, CRANE/IAA18, and others (Figure

2).

3. ARF7 and ARF19 regulate lateral root formation

via activation of LBD/ASL genes in Arabidopsis

To identify the genes regulated by ARF7, ARF19,

and SLR/IAA14 during LR initiation, the expression

profiles of wild-type, slr, and arf arf19 mutant roots

were analyzed (OKUSHIMA et al. 2005; VANNESTE et

al. 2005). Among the many kinds of auxin-responsive

genes containing AuxRE in their promoters, our group

focused on LBD16/ASL18 and LBD29/ ASL16 genes,

both of which encode nuclear proteins that belong to

the LBD (LATERAL ORGAN BOUNDARIES-DO-

MAIN)/ASL (AS2-LIKE) family (MATSUMURA et

al. 2009; SHUAI et al. 2002). Both LBD16/ASL18 and

LBD29/ASL16 genes are induced by auxin in roots

Fig. 1. LR formation is blocked in the slr and arf7 arf19

mutants in Arabidopsis.

Ten-day-old wild-type Columbia accession (WT),

slr-1/iaa14 (slr), and nph4-1 arf19-1 (arf7 arf19)

mutant plants are shown. After growth on standard

Murashige and Skoog medium, seedlings were

transferred to fresh medium for the photograph.

29GENETIC REGULATION OF LATERAL ROOT DEVELOPMENT IN ARABIDOPSIS

and are specifically expressed in the LR primordium

(OKUSHIMA et al. 2007). We have shown that over-

expression of either LBD16 or LBD29 partially res-

cues the arf7 arf19 defect in LR formation (Figure 3)

and that ARF7 and ARF19 directly activate the tran-

scription of LBD16/ASL18 and LBD29/ASL16 genes

(OKUSHIMA et al. 2007). In addition, overexpression of

LBD16-SRDX (LBD16 with a transcriptional repressor

domain) strongly suppresses LR formation, also sug-

gesting that endogenous LBD16 positively regulates

LR initiation (OKUSHIMA et al. 2007). These results in-

dicate that ARF7 and ARF19 regulate LR formation via

activation of LBD/ASL genes in Arabidopsis (Figure

2). The observation that a loss-of-function mutation of

LBD16 slightly decreases the number of LRs suggests

the redundant functions of LBD/ASL family members

in LR formation. Recently LBD18/ASL20 was shown

to regulate LR formation cooperatively with LBD16

(LEE et al. 2009). Further analysis of such LBD/ASL-

family proteins will be important for fully understand-

ing the mechanisms of auxin-mediated LR formation

in Arabidopsis. In rice, a mutation in the CRL1/ARL1

(CROWNROOTLESS1/ADVENTITIOUS ROOTLESS1)

gene, which encodes an LBD/ASL protein homologous

to Arabidopsis LBD29/ASL16, causes defects in ad-

ventitious root formation and LR formation (INUKAI et

al. 2005; LIU et al. 2005). These studies in Arabidop-

sis and monocots indicate that the basic mechanisms

of root formation are highly conserved between dicots

and monocots. Our group is now studying the redun-

dant functions of auxin-inducible LBD/ASL family

members and the LBD16-dependent signaling cascade

in LR formation.

4. Future perspectives

LR development is a powerful system for studying

important mechanisms in plant development, including

Fig. 2. Model of auxin-mediated LR initiation in Arabi-

dopsis.

Auxin signals captured by TIR1/AFBs auxin re-

ceptors promote the ubiquitination of Aux/IAAs

through the SCFTIR1/AFBs E3 ubiquitin ligase com-

plexes and degradation of Aux/IAAs by the 26S

proteasome. Degradation of Aux/IAAs (SLR/

IAA14 and the other IAAs) allows ARF7/19 to ac-

tivate LBD16/ASL18, LBD29/ASL16 and the other

target genes, thereby promoting LR initiation.

Fig. 3. Overexpression of LBD16 under the control of

the CaMV 35S promoter partially rescues the arf7

arf19 mutant phenotype in LR formation. Twelve-

day-old wild-type Columbia accession (WT), arf7

arf19, and 35S::LBD16/arf7 arf19 seedlings.

30 Hidehiro FUKAKI

meristem formation, lateral organ formation, plant hor-

mone signaling, cell fate specification, and cell cycle

regulation. Understanding of the molecular mecha-

nisms of LR development in Arabidopsis will allow us

to study not only the mechanisms of LR development

in other plant species (dicot/monocot plants and ferns)

but also the mechanism of the evolution of plant root

systems. Understanding the mechanisms of LR devel-

opment will also be helpful to produce transgenic crops

with a modified root architecture that easily adapts to

environmental stresses in the soil, such as water and

salinity stresses.

References

1. BARLOW, P.W., VOLKMANN, D., and BALUSKA, F. (2004).

Polarity in roots. In “Polarity in plants” (K. Lindsey, Ed),

pp. 192-241. Blackwell Publishing, Oxford.

2. CASIMIRO, I., MARCHANT, A., BHALERAO, R.P., BEECK-

MAN, T., DHOOGE, S., SWARUP, R., GRAHAM, N., INZE,

D., SANDBERG, G., CASERO, P.J., and BENNETT, M.

(2001). Auxin transport promotes Arabidopsis lateral

root development. Plant Cell 13: 43-852.

3. CASIMIRO, I., BEECKMAN, T., GRAHAM, N., BHALERAO,

R., ZHANG, H., CASERO, P., SANDBERG, G., and BEN-

NETT, M.J. (2003). Dissecting Arabidopsis lateral root

development. Trends Plant Sci. 8: 165-171.

4. DHARMASIRI, N., DHARMASIRI, S., and ESTELLE, M.

(2005). The F-box protein TIR1 is an auxin receptor.

Nature 435: 441-445.

5. FUKAKI, H., OKUSHIMA, Y. and TASAKA, M. (2007) Aux-

in-mediated lateral root formation in higher plants. Int.

Rev. Cytol. 256:111-137.

6. FUKAKI, H. and TASAKA M. (2009) Hormone interac-

tions during lateral root formation. Plant Mol. Biol. 69:

437-449.

7. FUKAKI, H., TAMEDA, S., MASUDA, H., and TASAKA, M.

(2002) Lateral root formation is blocked by a gain-of-

function mutation in the SOLITARY-ROOT/IAA14 gene

of Arabidopsis. Plant J. 29: 153-168.

8. FUKAKI, H., NAKAO, Y., OKUSHIMA, Y., THEOLOGIS, A.

and TASAKA, M. (2005) Tissue-specific expression of

stabilized SOLITARY-ROOT/IAA14 alters lateral root

development in Arabidopsis. Plant J. 44: 382-395.

9. HAGEN, G., and GUILFOYLE, T. (2002). Auxin-responsive

gene expression: genes, promoters and regulatory fac-

tors. Plant Mol. Biol. 49: 373-385.

10. INUKAI, Y., SAKAMOTO, T., UEGUCHI-TANAKA, M., SHI-

BATA, Y., GOMI, K., UMEMURA, I., HASEGAWA, Y., ASHI-

KARI, M., KITANO, H. and MATSUOKA. M. (2005) Crown

rootless1, which is essential for crown root formation in

rice, is a target of an AUXIN RESPONSE FACTOR in

auxin signaling. Plant Cell 17: 1387-1396.

11. KEPINSKI, S., and LEYSER, O. (2005). The Arabidopsis

TIR1 protein is an auxin receptor. Nature 435:446-451.

12. LASKOWSKI, M.J., WILLIAMS, M.E., NUSBAUM, H.C.,

and SUSSEX, I.M. (1995). Formation of lateral root meri-

stems is a two-stage process. Development 121: 3303-

3310.

13. LEE, H.W., KIM, N.Y., LEE, D.J., and KIM, J. (2009)

LBD18/ASL20 regulates lateral root formation in com-

bination with LBD16/ASL18 downstream of ARF7 and

ARF19 in Arabidopsis. Plant Physiol. 151: 1377-1389.

14. LIU, H., WANG, S., YU, X., YU, J., HE, X., ZHANG, S.,

SHOU, H., and WU, P. (2005). ARL1, a LOB-domain

protein required for adventitious root formation in rice.

Plant J. 43: 47-56.

15. MALAMY, J.E., and BENFEY, P.N. (1997). Organization

and cell differentiation in lateral roots of Arabidopsis

thaliana. Development 124: 33-44.

16. MALAMY, J.E. (2005) Intrinsic and environmental re-

sponse pathways that regulate root system architecture.

Plant Cell Environ. 28: 67-77.

17. MATSUMURA, Y., IWAKAWA, H., MACHIDA, Y., and MACHI-

DA, C. (2009) Characterization of genes in the ASYM-

METRIC LEAVES2/LATERAL ORGAN BOUNDAR-

IES (AS2/LOB) family in Arabidopsis thaliana, and

functional and molecular comparisons between AS2 and

other family members. Plant J. 58: 525-537.

18. MOCKAITIS, K. and ESTELLE, M. (2008) Auxin recep-

tors and plant development: A new signaling paradigm.

Annu. Rev. Cell Dev. Biol. 24: 55-80.

19. OKUSHIMA, Y., FUKAKI, H., ONODA, M., THEOLOGIS,

A., and TASAKA, M. (2007) ARF7 and ARF19 regulate

lateral root formation via direct activation of LBD/ASL

genes in Arabidopsis. Plant Cell 19: 118-130.

20. OKUSHIMA, Y., OVERVOORDE, P.J., ARIMA, K., ALONSO,

J.M., CHAN, A., CHANG, C., ECKER, J.R., HUGHES, B.,

LUI, A., NGUYEN, D., ONODERA, C., QUACH, H., SMITH,

A., YU, G., and THEOLOGIS, A. (2005) Functional ge-

nomic analysis of the AUXIN RESPONSE FACTOR

gene family members in Arabidopsis thaliana: unique

and overlapping functions of ARF7 and ARF19. Plant

Cell 17: 444-463.

21. REED, R.C., BRADY, S.R., and MUDAY, G.R. (1998). In-

hibition of auxin movement from the shoot into the root


inhibits lateral root development in Arabidopsis. Plant

Physiol. 118: 1369-1378.

22. SHUAI, B., REYNAGA-PENA, C.G., and SPRINGER, P.S.

(2002). The LATERAL ORGAN BOUNDARIES gene de-

fines a novel, plant-specific gene family. Plant Physiol.

129: 747-761.

23. UEHARA, T., OKUSHIMA, Y., MIMURA, T., TASAKA, M.

and FUKAKI, H. (2008) Domain II mutations in CRANE/

IAA18 suppress lateral root formation and affect shoot

development in Arabidopsis thaliana. Plant Cell Physi-

ol. 49: 1025-1038.

24. VANNESTE, S., RYBEL, B.D., BEEMSTER, G.T.S., LJUNG,

K., SMET, I.D, VAN ISTERDAEL, G., NAUDTS, M., IIDA,

R., GRUISSEM, W., TASAKA, M., INZÉ, M., FUKAKI, H.

and BEECKMAN, T. (2005) Cell cycle progression in the

pericycle is not sufficient for SOLITARY-ROOT/IAA14-

mediated lateral root initiation in Arabidopsis thaliana.

Plant Cell 17: 3035-3050.

25. WILMOTH, J.C., WANG, S., TIWARI, S.B., JOSHI, A.D.,

HAGEN, G., GUILFOYLE, T.J., ALONSO, J.M., ECKER, J.R.,

and REED, J. (2005). NPH4/ARF7 and ARF19 promote

leaf expansion and auxin-induced lateral root formation.

Plant J. 43: 118-130.

32 Hidehiro FUKAKI

　維管束植物の地下部を構成する根系は，地上部を支持し，土壌から水分や養分を吸収する上で重要な役割を果たす。根系は発芽後伸長する主根と，主根から形成される側根，地上部から形成される不定根によって構築される。古くから植物ホルモンのオーキシンが側根形成や不定根形成に重要な役割を果たすことが知られていたが，その分子機構は未だに解明されていない。我々の研究グループは，オーキシンを介した側根形成の分子機構に注目して，これまで側根形成能に欠損をもつシロイヌナズナ slr（solitary-root）変異体，および arf7 arf19二重変異体の解析から，オーキシン応答転写因子 ARF7, ARF19と Aux/IAAタンパク質 SLR/IAA14との相互作用を介した遺伝子発現制御が側根形成開始に重要なことを明らかにしてきた。そして，マイクロアレイ解析などからARF7, ARF19の標的遺伝子として植物に特有なLBD (Lateral Organ Boundaries-domain)/ASL (AS2-

Like)ファミリーに属する LBD16/ASL18と LBD29/

ASL16を同定し，これらが側根形成開始で機能することを明らかにした。現在，側根形成におけるオーキシン誘導性 LBD/ASLタンパク質群の機能と，LBD16を介した分子カスケードの解明を目指して研究を進めている。　側根発生は，分裂組織形成や器官形成，植物ホルモンのシグナル伝達，細胞運命の決定機構，細胞周期制御といった植物科学における重要な問題を研究する上で良いモデルといえる。側根発生の分子機構の解明は，他の植物種の側根発生機構の理解に役立つだけでなく，植物界における根系の進化のしくみを解く手がかりとなる。また，将来，さまざまな環境ストレス下において作物の収量を維持・増加させるような植物を作出する際に，根系パターンの改変技術が有用となると考えられる。

シロイヌナズナにおける側根発生の遺伝的制御機構

深　城　英　弘

神戸大学大学院理学研究科生物学専攻


質疑応答西澤：側根形成に関わる可能性のある遺伝子リストにジベレリン関連の遺伝子があったと思うのですが，側根形成にジベレリンが関与しているのでしょうか。深城：側根形成にジベレリンが関与しているかどうかに関して，私たちは直接的なデータは持っていません。側根形成と植物ホルモンに関する総説（FUKAKI and TASAKA, Plant Mol. Biol.,

2009）にも書きましたが，根の形成におけるジベレリンの役割に関する文献はとても少ないです。根の伸長に関するジベレリンの役割は知られていますが，側根形成については，あまり詳しく調べられていないだけで，ジベレリン関係の突然変異体を使って丁寧に調べると何か表現型が見つかるかもしれません。廣近：arf7 arf19二重変異体で LBD16を 35Sプロモーターでユビキタスに過剰発現させた場合，側根が無数にできない理由というのは，何か位置情報を認識しているということなのでしょうか。深城：arf7 arf19二重変異体における LBD16の過

剰発現体では，側根数は野生型よりも少ないですし，至るところから側根が出るわけではありません。この形質転換ラインは LBD16をかなり高いレベルで過剰発現していますが，ここまでしか回復しないということは，逆に，側根を抑える仕組みがあるのかもしれません。野生型でも側根ができると，すぐそばに側根はできません。これは動物の発生で言われている lateral

inhibitionと同じメカニズムで，いったん原基ができると，そのすぐ両側には作らない仕組みがあると考えられます。LBD16の過剰発現の影響があったとしても，逆にそれを抑え込む仕組みが arf７arf19二重変異体に残っている可能性を示唆しています。廣近：側根形成頻度が高くなる変異体はないですか。深城：側根形成頻度が高くなる変異体として，オーキシンの過剰合成変異体が知られています。それ以外でも側根形成頻度が高くなる変異体は，いくつか知られているので，側根頻度を抑制する遺伝子座があると思います。

35

Introduction

Although it is known that roots play important

roles in the adaptation of crop plants to stresses such

as drought (WANG and YAMAUCHI 2006), the specific

root traits responsible for this adaptation depend on the

intensity, duration, and nature of the drought. For ex-

ample, under upland conditions where soil moisture is

available mainly in the deep layer, the desirable trait

may be long, thick roots. On the other hand, under

typical field conditions, the soil environment changes

throughout the cropping season and can be heteroge-

neous even at a particular depth.

In the case of rice, more than half of the area plant-

ed consists of rainfed lowland fields without irrigation.

The most severe constraint to production in these areas

is water stress. Under rainfed lowland conditions, a wa-

ter-impermeable hardpan tends to form in soils with a

significant clay content approximately 20 cm below the

soil surface, and thus waterlogged conditions can tem-

porarily appear. In this situation, the subsoil below the

hardpan is usually wet while the topsoil above the hard-

pan is often exposed to frequent wet/dry cycles that are

caused by irregular rainfall, causing substantial reduc-

tion in growth and yield of rice plants. Because of this

variability, the water stress experienced under rainfed

lowland conditions is qualitatively different from the

more uniform drought stress experienced under upland

conditions (WADE et al. 1999). Furthermore, the global

trend of increasing water scarcity urgently demands the

development of water-saving rice production technol-

ogy in irrigated rice fields, which experience a type of

water stress similar to that in rainfed lowlands: alterna-

tion between anaerobic, flooded conditions and aero-

bic, droughted conditions.

In this review, we evaluated the genetic variation

reported in dry matter production, shoot growth, and

root system development in response to drought to

identify key root traits that contribute to plant adapta-

tion to various intensities of water stress. In addition,

we tried to determine the contribution of plastic devel-

opment (defined below) and associated physiological

responses of roots to shoot dry matter production under

conditions of fluctuating soil moisture stress.

Identification of key root traits for adaptation to

various intensities of water stress

The root system of an individual rice plant consists

of different component roots. These component roots

differ in morphology, anatomy, and physiological func-

tion, and in developmental responses to various environ-

ments. The ability of plants to change developmentally

and functionally to survive and continue growing under

various environmental conditions is termed phenotypic

plasticity (O’ TOOLE and BLAND, 1987). YAMAUCHI et

al. (1996) indicated that phenotypic plasticity in root

system morphology is a key trait for the stress tolerance

of crop plants. Recently, we showed, using hydropon-

ics experiments with young seedlings, that the plastic

development of different component roots to drought is

controlled by different QTLs (WANG et al. 2005).

The importance of root plasticity was evaluated

in field experiments conducted at the experimental


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

Yoshiaki INUKAI

Graduate School of Bioagricultural Sciences, Nagoya University

Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

36 Yoshiaki INUKAI

farm of Nagoya University, Nagoya, Japan in 2005

and 2006. We used a set of 54 chromosome segment

substitution lines (CSSLs) derived from Nipponbare

(japonica type) and Kasalath (indica type); these were

provided by the Rice Genome Research Center of the

National Institute of Agrobiological Sciences, Japan

(KANOU et al. 2007). Each line has one or a few defined

chromosome segments of the Kasalath genome in the

genetic background of Nipponbare. This set of CSSLs

is a powerful tool for identifying desirable root traits

under water stress conditions as well as for evaluating

the functional significance of the target traits with mini-

mal confounding effects of other traits. Those CSSLs,

together with their parents (Nipponbare and Kasalath)

and KDML105 (a rainfed lowland rice variety from

Thailand) were grown in a watertight experimental bed

equipped with a line-source sprinkler system that can

create various intensities of drought stress. The volu-

metric soil moisture content (SMC) was monitored with

a soil moisture sensor (Em 50, Decagon, Logan, Utah,

USA). This system successfully created and maintained

a soil moisture gradient with an SMC range of about

50% to 10% (equivalent to water potential of 0 MPa to

-0.098 MPa).

In both years, most of the CSSLs showed reduced

plant height, tiller production, and dry matter produc-

tion as drought intensified. Such reductions were asso-

ciated with reduced photosynthesis, transpiration, and

stomatal conductance. Nipponbare showed the most

severely reduced dry matter production among all the

genotypes examined as the drought intensified under

field conditions. In contrast, some lines showed differ-

ent responses from Nipponbare. CSSL45 and CSSL50

maintained relatively high dry matter production under

conditions of increasing drought stress. These lines

also tended to maintain or promote root elongation and

branching under the same conditions. Specifically, the

shoot and root growth of CSSL45 and CSSL50 were

not different from those of Nipponbare at SMC of 40%

and above, whereas the dry matter production and total

root length of those CSSLs peaked at SMC of around

30–35%. Under severe drought conditions (SMC of

10% or below), the extent of shoot and root growth re-

duction was similar to that of Nipponbare (KANOU et

al. 2007).

In addition, dry matter production in KDML105,

increased as drought stress intensified, and the root sys-

tem development (evaluated based on total root length)

showed the same trend as did the shoot growth. Such

plastic root responses were found to be the result of

a combination of increased tiller production and in-

creased nodal root development from the tillers (i.e.,

increased number, elongation, and branching) (KANOU

et al. 2007). These results strongly suggest that plastic

root responses of rice genotypes may be one of the key

traits that contribute to adaptation to various intensities

of water stress.

Contribution of plastic development and associated

physiological responses of roots to shoot dry matter

production under fluctuating soil moisture stress

Drought and waterlogging are two of the most

important abiotic stresses affecting plant growth and

development. For this reason, it is important to under-

stand that these soil moisture stresses transiently recur

in the field due to the intermittent nature of rainfall pat-

terns and irrigation systems. Hence, the soil is exposed

to frequent episodes and varying degrees of alternate

dry and wet conditions, causing transient drought alter-

nating with O2 deficiency. The inability of the plant to

acclimate to such changes may result in reduced growth

and dry matter production. However, most studies have

dealt only with the effects of one or the other of these

stresses (WANG and YAMAUCHI 2006). Little attention

has been devoted to their interactive effects.

Previous studies have provided evidence that

changing soil moisture conditions adversely affect crop

root development mainly by reducing root elongation

and branching into lateral roots. These limit soil water

uptake ability, which then results in reduced stomatal

conductance, transpiration, and photosynthesis. Ul-

timately, this results in reduced shoot dry matter pro-

duction (BAÑOC et al. 2000a, b; PARDALES and YAM-

AUCHI 2003; SIOPONGCO et al. 2008, 2009; SUBERE et

al. 2009; WADE et al. 2000; YAMAUCHI et al. 1996).

37RICE ROOT IMPROVEMENT FOR ADAPTATION TO WATER STRESS

Cognizant of this, several authors have proposed that

the ability of roots to respond to these fluctuating soil

moisture regimes is one of the important developmental

traits for adaptation (INGRAM et al. 1994; WANG and

YAMAUCHI 2006; YAMAUCHI et al. 1996). Under pro-

gressive soil drying, root responses include increased

root length density (SIOPONGCO et al. 2005) as a re-

sult of plastic lateral root development (AZHIRI-SIG-

ARI et al. 2000; BAÑOC et al. 2000b; KAMOSHITA et al.

2000). On the other hand, plants having shallow root

systems with enhanced aerenchyma that provide a less

resistant pathway for internal atmospheric O2 diffusion

to the root tips are better adapted to sudden waterlog-

ging (COLMER 2003; JUSTIN and ARMSTRONG 1987;

SURALTA and YAMAUCHI 2008). Under fluctuating soil

moisture levels, rice plants develop root systems that

are readily adaptable to changing stresses, for example,

increased root elongation rate and plastic lateral root

production under dry conditions (BAÑOC et al. 2000b)

and enhanced aerenchyma under suddenly waterlogged

conditions (JUSTIN and ARMSTRONG 1987). In our study

of rice (SURALTA et al. 2008a), we found that aerobic

rice cultivars, high-yielding rice grown in non-puddled,

aerobic soils under irrigation and high external inputs,

have greater ability for plastic lateral root production

than irrigated lowland cultivars under fluctuating mois-

ture stresses. Specifically, the greater ability for plastic

lateral root production in the aerobic cultivars was due

to their ability to maintain a higher rate of seminal root

elongation and to develop more nodal and lateral roots

in response to a switch from O2 deficiency to droughted

conditions, and a higher rate of seminal root elongation,

more aerenchyma formation in the seminal root axis,

and a greater number of nodal roots in response to a

switch from droughted to O2-deficient conditions.

To minimize the effects of genetic confounding

and increase the precision of the analysis, the above

root-trait responses were validated using the previously

described CSSLs of Nipponbare carrying chromosome

segments of Kasalath (SURALTA et al. 2008b). We iden-

tified one line (CSSL47) that had no significant differ-

ence in shoot and root growth from Nipponbare under

non-stressed (continuously well-aerated) conditions.

CSSL47, however, consistently showed more plastic

root development than Nipponbare, in terms of more

branching of lateral roots and more aerenchyma forma-

tion under transient moisture stresses in hydroponics

such as O2-deficient-to-droughted and vice-versa. In-

terestingly, the plastic root development responses of

CSSL47 did not contribute to greater shoot dry matter

production under non-stressed conditions. We therefore

attempted to test the hypothesis that plastic responses

in root traits are useful in maintaining crop productiv-

ity if plants are grown under conditions of fluctuating

soil moisture stress (SURALTA et al. 2010). We exam-

ined root plastic development (aerenchyma formation

and lateral root production) and the ability of the root

system to maintain atmospheric O2 transport and soil

water uptake under fluctuating soil moisture stresses.

The root responses were also quantified in terms of

their contribution to the maintenance of physiological

responses such as stomatal conductance, transpiration,

and photosynthesis and ultimately, shoot dry matter

production. To increase the precision of the quantitative

analysis, we used CSSL47 and compared it with the

recurrent parent Nipponbare to minimize the effect of

genetic confounding.

We have precisely quantified that the more plas-

tic root development of CSSL47 and associated physi-

ological functions under transient soil moisture stress

contributed to improved maintenance of shoot dry mat-

ter production of CSSL47 when compared with Nip-

ponbare (SURALTA et al. 2010). Specifically, under the

drought-to-waterlogged (D–W) condition, root plastic-

ity was expressed as aerenchyma formation. CSSL47

showed greater shoot dry matter production than Nip-

ponbare due to the higher rate of recovery of CSSL47

in terms of stomatal conductance, transpiration, and

photosynthesis in response to waterlogging after a pe-

riod of drought. This recovery was supported by the

greater root system development of CSSL47, owing to

its greater ability to enhance the aerenchyma system to

facilitate O2 diffusion to the root tips under the D–W

condition. On the other hand, under the waterlogged-

to-droughted (W–D) condition, the root plasticity was

expressed as lateral root branching. CSSL47 showed

38 Yoshiaki INUKAI

greater shoot dry matter production than Nipponbare

due to its greater ability to maintain stomatal conduc-

tance, transpiration, and photosynthesis. This increased

ability to maintain stomatal conductance, transpiration,

and photosynthesis was attributed to CSSL47’s greater

root system development due to increased branching,

which facilitated higher water extraction during drought

after waterlogging (W–D condition). Detailed analyses

of plastic root developmental responses, especially

from the upper nodes, to different timing of fluctuat-

ing water stresses and different stages of growth (i.e.,

vegetative and reproductive stage) are ongoing. This is

being done to validate the plastic root responses found

at the seedling stage in the present study and to quantify

their contribution to the maintenance of shoot dry mat-

ter production and grain yield.

Acknowledgement

This work was supported by a grant from the Min-

istry of Agriculture, Forestry and Fisheries of Japan

(Genomics for Agricultural Innovation, QTL-4004).

References

1. AZHIRI-SIGARI, T., YAMAUCHI, A., KAMOSHITA, A., and

WADE, L. J. (2000). Genotypic variation in response of

rainfed lowland rice to drought and rewatering. II. Root

growth. Plant Prod. Sci. 3: 180-188.

2. BAÑOC, D. M., YAMAUCH, A., KAMOSHITA, A., WADE, L.

J., and PARDALES, J. R. Jr. (2000a). Genotypic variations

in response of lateral root development to fluctuating

soil moisture in rice. Plant Prod. Sci. 3: 335-343.

3. BAÑOC, D. M., YAMAUCH, A., KAMOSHITA, A., WADE, L.

J., and PARDALES, J. R. Jr. (2000b). Genotypic variations

in response of lateral root development to fluctuating

soil moisture in rice. Plant Prod. Sci. 3: 335-343.

4. COLMER, T. D. (2003). Aerenchyma and an inducible

barrier to radial oxygen loss facilitate root aeration in

upland, paddy and deepwater rice (Oryza sativa L.).

Ann. Bot. 91: 301-309.

5. INGRAM, K. T., BUENO, F. D., NAMUCO, O. S., YAMBAO,

E. B. and BEYROURY, C. A. (1994). Rice root traits for

drought resistance and their genetic variation. InRice

roots: nutrient and water use (ed. Kirk, C. J. D.). Interna-

tional Rice Research Institute, Manila, pp 66-67.

6. JUSTIN, S. H. F. W. and ARMSTRONG, W. (1987). The ana-

tomical characteristics of roots and plant response to soil

flooding. New Phytol. 106: 465-495.

7. KAMOSHITA, A., WADE, L. J. and YAMAUCHI, A. (2000).

Genotypic variation in response of rainfed lowland rice

to drought and rewatering. III. Water extraction during

the drought period. Plant Prod. Sci. 3:189-196.

8. KANOU, M., INUKAI, Y., KITANO, H. and YAMAUCHI, A.

(2007). Identification of key root traits for adaptation of

rice genotypes to various intensities of water stress. Pro-

ceedings of the 2nd International Conference on Rice for

the Future: 216-220.

9. O’TOOLE, J. C. and BLAND, W. L. (1987). Genotypic

variation in crop plant root systems. Advance in Agron-

omy. 41: 91-145.

10. PARDALES, J. R. Jr. and YAMAUCHI, A. (2003). Regula-

tion of root development in sweetpotato and cassava by

soil moisture during their establishment period. Plant

Soil 255: 201-208.

11. SIOPONGCO, J. D. L. C., YAMAUCHI, A., SALEKDEH, H.,

BENNETT, J. and WADE, L. J. (2005). Root growth and

water extraction responses of doubled-haploid rice lines

to drought and rewatering during the vegetative stage.

Plant Prod. Sci. 8: 497-508.

12. SIOPONGCO, J. D. L. C., SEKIYA, K., YAMAUCHI, A.,

EGDANE, J., ISMAIL, A. M. and WADE, L. J. (2008). Sto-

matal responses in rainfed lowland rice to partial soil

drying; evidence of root signals. Plant Prod. Sci. 11: 28-

41.

13. SIOPONGCO, J. D. L. C., SEKIYA, K., YAMAUCHI, A.,

EGDANE, J., ISMAIL, A. M. and WADE, L. J. (2009). Sto-

matal responses in rainfed lowland rice to partial soil

drying; comparison of two lines. Plant Prod. Sci. 12: 17-

28.

14. SUBERE, J. O. Q., BOLATETE, D., BERGANTIN, R., PAR-

DALES, A., BELMONTE, J. J., MARISCAL, A., SEBIDOS,

R. and YAMAUCHI, A. (2009). Genotypic variation in

responses of cassava (Manihot esculenta Crantz) to

drought and rewatering. I. Root system development.


15. SURALTA, R. R., INUKAI, Y. and YAMAUCHI, A. (2008a)

Genotypic variations in responses of lateral root devel-

opment to transient moisture stresses in rice cultivars.


16. SURALTA, R. R., INUKAI, Y. and YAMAUCHI, A. (2008b).

Utilizing chromosome segment substitution lines

(CSSLs) for evaluation of root responses under transient

moisture stresses in rice. Plant Prod. Sci. 11: 457-465.

17. SURALTA, R. R., INUKAI, Y. and YAMAUCHI, A. (2010).

Dry matter production in relation to root plastic develop-

ment, oxygen transport, and water uptake of rice under


transient soil moisture stresses. Plant Soil 332: 87-104.

18. SURALTA, R. R., and YAMAUCHI, A. (2008). Root growth,

aerenchyma development, and oxygen transport in rice

genotypes subjected to drought and waterlogging. Envi-

ron. Exp. Bot. 64: 75-82.

19. WADE, L. J., KAMOSHITA, A., YAMAUCHI, A. and AZHIRI-

SIGARI, T. (2000). Genotypic variation in response of

rainfed lowland rice to drought and rewatering I. Growth

and water use. Plant Prod. Sci. 3: 173-179.

20. WADE, L. J., FUKAI, S., SAMSON, B. K., ALI, A. and MA-

ZID, M. A. (1999). Rainfed lowland rice: Physical envi-

ronment and cultivar requirements. Field Crops Res. 64:

3-12.

21. WANG, H. and YAMAUCHI, A. (2006). Growth and Func-

tion of Roots under Abiotic Stress in Soil. In Plant-Envi-

ronment Interactions (3rd) (ed. Huang, B.). CRC Press,

New York. pp. 271-320.

22. WANG, H., INUKAI, Y., KAMOSHITA, A., WADE, L. J.,

SIOPONGCO, J. D. L. C., NGUYEN, H. and YAMAUCHI,

A. (2005). QTL analysis on plasticity in lateral root de-

velopment in response to water stress in the rice plant. In

Rice is life: scientific perspectives for the 21st century.

The Proceedings of the World Rice Research Confer-

ence. (eds. Toriyama, K. et al.). IRRI, Los Baños and

JIRCAS, Tsukuba. pp. 464-469.

23. YAMAUCHI, A., PARDALES, J. R. Jr. and KONO, Y. (1996).

40 Yoshiaki INUKAI

　天水田における水ストレスは，畑状態で起こる乾燥ストレスとは質的に異なる。天水田には深さ 20cmあたりに硬盤層が存在し，これにより根の下層への伸長が抑制される。硬盤層は不透水層であるため，これより下の心土は通常，湿潤状態であるが，硬盤層より上の作土は不定期な降雨によって嫌気的と好気的条件を繰り返し，その際に起こる土壌水分変動ストレスが生産性の低下を招く。本総説では，このような栽培環境下での水ストレスを回避する上で重要な根系形質について提案したい。

＜乾燥ストレス下で重要な根系形質＞　我々は，湿潤から乾燥まで連続的な土壌水分勾配を発生させることを可能にする Line source

sprinkler 法により，様々な品種を評価した。その結果，湿潤状態での根系に比べ，低土壌水分状態において著しく根系を発達させる能力を有する品種，すなわち根系の可塑的反応性の優れた品種が非常に高い耐旱性を有することが判明した。これらの品種の中には，実際に乾燥ストレスが問題となる東北タイの天水田地域において長年にわたり作付け面積第一位を誇っている KDML105も含まれていた。　一方，地上部形態の大きく異なる品種間において耐旱性を比べる上では，根系の可塑的反応が実際にどの程度耐旱性に貢献しているのかを判断するのは困難である。そこで，基本的な遺伝背景は日本晴であるが，一部の染色体断片が Kasalath

由来の断片に置換されている染色体断片置換系統群（CSSL，農業生物資源研究所イネゲノムリ

ソースセンターより分譲）54系統を用い，日本晴との間において耐旱性能力を評価した結果，低土壌水分下での地上部成育の維持能力が最も高い系統として CSSL50が選抜された。湿潤条件下における地上部成育，および根系発育には，日本晴と CSSL50との間で優位な差は見られなかったが，とくに土壌含水率が 20％以下の低土壌水分ストレス下では CSSL50の総根長は日本晴の約 2

倍にまで達していた。また，ストレス下での総根長と積算蒸散量との間には有意な正の相関が認められ，以上より土壌乾燥ストレス条件に適応できるイネは，低土壌水分条件下において根系が可塑的に反応することによって高い養水分吸収が可能となり，その結果として乾物生産が維持できると考えられる。

＜�乾燥後の降雨による嫌気ストレス下で重要な根系形質＞　加えて，上述の KDML105は，乾燥状態から再灌水を行った場合における蒸散の回復能力が他品種よりも優れていることが判明している。また我々は，日本晴 / Kasalathの CSSL47は，日本晴に比べて乾燥土壌で成育した根が湛水条件下に移された時に，根端への酸素輸送速度を規定する通気組織を形成する能力が高く，これにより根系発達が促進され，気孔コンダクタンス，蒸散速度，および光合成速度の回復が早いことを見出した。従って，著しく土壌水分が変動する天水田での安定生産を目指す上では，このような嫌気ストレス応答に関わる有用遺伝子座についても注目し，品種改良を進めることが重要となろう。

耐旱性向上を目指したイネ根系形質の遺伝的改良

犬　飼　義　明

名古屋大学大学院生命農学研究科


質疑応答司会：犬飼先生，どうもありがとうございました。生物をやってる者は乾燥ストレスとかいうと，すぐ DREBとかを，細胞レベルの話をイメージしますが，きょうのお話は自然界というかまあ，栽培植物ですけれども，そういったものではこういったその個体レベルでの応答みたいのは非常に重要だってことのお話だったと思います。それではご質問，ご意見ございませんでしょうか。久保山：茨城大学の久保山です。先ほど，染色体置換系統を使って QTLの結局ポジショナルクローニングというか，そういうストラテジーだったんですけども，その前の話が KDML105

だったんですけども，Kasalathは，どのぐらい根に関して日本晴と比べて差があるということなんでしょう。犬飼：日本晴に比べては，根っこ，総根長みたいなものは土壌水分は低下しても，ある程度維持されるっていう傾向があります。KDMLのようにぶわっと増えるっていうことはないんです

けども，ある程度日本晴に比べたら，Kasalath

の根っこの長さっていうのは，ストレス下でも維持されるって傾向があります。また，日本晴がこう簡単にやられるのに対して，Kasalathはある程度は地上部の成長を維持できるので，日本晴に比べたら根の発達はいい方だと思います。久保山：最終的な総合性検定みたいな実験では，

KDMLのアレソック候補遺伝子を入れるような形で，検定するとかそういうようなお考えはありますか？犬飼：そうですね。日本晴に対して KDML側の遺伝子を入れて，側根の発達が良くなるとかっていうのを見ていきたいと思います。久保山：ありがとうございます。司会：時間きているようですが，もう１つぐらい質問，ございませんでしょうか。よろしいでしょうか。じゃ，時間のきておりますので，犬飼先生，どうもありがとうございました。

（拍手）

43

Introduction

As the current increase in greenhouse gases is an-

ticipated to continue, the period of high-temperature

stress encountered by crops during the growing season

is expected to be prolonged, and this may have a sig-

nificant impact on crop production. The effects of in-

creasing temperatures and high atmospheric CO2 con-

centrations on rice yield have been analyzed using crop

simulation models (HORIe et al. 1996, NAKAGAWA et al.

2003) and studies under a controlled environment (HO-

RIE et al. 1995, KIM et al. 1996, MATSUI et al. 1997).

The high temperatures that are anticipated have been

shown to increase floret sterility and decrease rice yield

(HORIE et al. 1996, NAKAGAWA et al. 2003). Under-

standing the conditions that induce floret sterility and

the plant characteristics that determine tolerance of

these conditions would help researchers to predict the

effects of climate change on rice production and to de-

velop countermeasures against yield losses that might

result from high temperatures. Here, we review studies

related to the occurrence of heat-induced floret sterility

(HIFS) and to the morphological markers that deter-

mine tolerance of high temperatures, including results

from our recent unpublished studies.

Occurrence of HIFS in the field

Previous experiments in controlled-environment

chambers have shown that temperatures greater than 34

°C (SATAKE and YOSHIDA 1978) or 35 °C (MATSUI et

al. 2001) at the time of anthesis induced floret sterility.

These threshold temperatures coincide with the tempera-

ture ranges that have induced floret sterility in field-grown

rice in Thailand (OSADA et al. 1973). Based on these data,

some scientists have predicted that HIFS during flower-

ing can decrease rice yield and may become a serious

problem as a result of global warming, even in temperate

regions such as Japan (HORIE et al. 1996, NAKAGAWA et

al. 2003). On the other hand, Angus (1997) reported that

the maximum daily air temperature during the anthesis

period occasionally reaches 40 ºC in paddy fields in Aus-

tralia with no serious yield losses due to HIFS.

Our field observations showed that strong transpi-

rational cooling of the rice canopy due to a high va-

por-pressure deficit and strong winds on clear days can

reduce the panicle temperature by as much as 6.8 ºC

compared with the ambient temperature (MATSUI et al.

2007). In contrast, the panicle temperature under the

humid and calm conditions found at Yangtze Univer-

sity in Jingzhou, China, was higher than the air tem-

perature (TIAN et al., 2010). The factors that regulate

panicle temperature therefore appear to be important in

explaining HIFS.

Mechanisms responsible for HIFS

The mechanisms responsible for HIFS have been


Heat-induced Floret Sterility in Rice:Mechanisms of Occurrence and Tolerance

Tsutomu MATSUI1, Norvie L. MANIGBAS2, Edilberto REDOÑA3, Xiaohai TIAN4,

Mayumi YOSHIMOTO5 and Toshihiro HASEGAWA5

1Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan2Philippine Rice Research Institute, Maligaya, Science City of Muñoz, Nueva Ecija 3119, Philippines3International Rice Research Institute, Los Baños, Laguna, Philippines4Faculty of Agriculture, Yangtze University, Jingzhou, Hubei 434025, China5National Institute for Agro-Environmental Sciences, 3-1-3, Kannondai, Tsukuba, Ibaraki 305-8604, Japan

44 Tsutomu MATSUI, Norvie L. MANIGBAS, Edilberto REDOÑA, Xiaohai TIAN, Mayumi YOSHIMOTO and Toshihiro HASEGAWA

studied using controlled-environment systems such

as phytotrons, temperature-gradient chambers, and

growth chambers. The direct cause of the sterility was

found to be a reduction in the number of pollen grains

that germinated on the stigma (SATAKE and YOSHIDA

1978, MATSUI et al. 2001). Decreases in either the to-

tal number of pollen grains that arrive on the stigma

or the percentage of these grains that germinate can

reduce the number of germinated pollen grains on the

stigma, but in many cases, the decrease in total number

was the main cause of sterility (SATAKE and YOSHIDA

1978, MATSUI et al. 2001). The difference in tolerance

of HIFS among cultivars has also been found to depend

on variations in the stability of pollination under high-

temperature conditions (SATAKE and YOSHIDA 1978,

MATSUI et al. 2001).

The driving force responsible for anther dehiscence

(Fig. 1) is rapid swelling of pollen grains in the loc-

ule in response to floret opening (MATSUI et al. 1999).

High temperatures during flowering inhibit the swell-

ing of pollen grains, resulting in anther indehiscence

(MATSUI et al. 2000) and poor release of pollen grains

(MATSUI et al. 2005). Under severe high-temperature

conditions, this inhibition results in indehiscence (MAT-

SUI et al. 2000). Under normal high-temperature condi-

tions, the inhibition of swelling is not enough to cause

indehiscence but does seem to cause failure of pollen

release (MATSUI 2005). High temperatures at flowering

make pollen grains sticky (SATAKE and YOSHIDA 1978,

MATSUI 2005), and sticky grains can block the basal

dehiscence and remain in the anther (MATSUI 2005).

Marker for screening rice cultivars tolerant of high

temperatures at flowering

1. The role of basal dehiscence in stable pollination

To improve the tolerance of rice to high tempera-

tures, it is important to identify the traits that control or

represent the tolerance of pollination to high tempera-

tures at flowering. MATSUI and KAGATA (2003) found

that the length of the basal dehiscence was positively

correlated with the stability of pollination under nor-

mal conditions (Fig. 2). Moreover, MATSUI et al. (2005)

Fig. 1. Diversity of dehisced anthers in old Japanese cul-

tivars. Long basal dehiscence enhances stable self-

pollination. Cultivars, from left to right: 1, Mub-

ouaikoku; 2, Ginbouzu; 3, Homura; 4, Magatama

(MATSUI 2009).

Fig. 2. Schematic illustration of the dispersal of pollen

from anthers with (A) short basal pores and (B)

long basal pores. Most of the pollen grains in the

anthers with short basal pores remain in the the-

cae until the anther bends down. In contrast, most

of the pollen grains in the anthers with long basal

pores drop from the pores at the beginning of floret

opening, which coincides with the beginning of an-

ther dehiscence (MATSUI and KAGATA 2003).

45HEAT-INDUCED FLORET STERILITY IN RICE

found that the length of the basal dehiscence was

strongly and positively correlated with the percentage

of sufficiently pollinated florets under hot and humid

conditions during flowering in a growth chamber (Fig.

3). This measurement explained 95% of the variance in

the percentage of sufficiently pollinated florets, which

ranged from 5 to 85% among 18 cultivars under hot

and humid conditions (MATSUI et al. 2005). Therefore,

a long basal dehiscence increases the reliability of pol-

lination under high-temperature conditions, probably

through the improved ease of pollen transport from the

dehisced anther to the stigma (MATSUI and KAGATA

2003). MATSUI et al. (2005) also found that the length

of the basal dehiscence under normal conditions was

closely and significantly positively correlated with that

under high-temperature conditions (Fig. 4). We can

therefore predict the tolerance of pollination for high

temperatures by measuring the length of the basal de-

hiscence under normal conditions. A long basal dehis-

cence may be a useful marker for screening rice culti-

vars capable of tolerating high temperatures.

2. Field performance of long anther dehiscence as a

marker for screening cultivars capable of tolerat-

ing high temperatures

Case 1: Yangtze Valley, China

Serious yield losses due to floret sterility occurred

in 2003, when the hottest summer temperatures in re-

corded history affected the reproductive stage of the

rice crop in China’s Yangtze Valley, where HIFS has

occasionally been observed in paddy fields. Practical

differences in seed set rates among cultivars have been

reported (MATSUI 2009). To confirm whether a simple

marker (long basal dehiscence) could be used in the

field, we examined the relationship between the length

of the basal dehiscence and fertility under hot and hu-

mid field conditions in Jingzhou (at Yangtze Universi-

ty), along the middle reaches of the Yangtze River.

The experiment was conducted during the sum-

Fig. 3. Relationship between the length of the dehiscence

at the basal part of the thecae and the percentage

of florets bearing more than 20 pollen grains under

conditions of high temperature and humidity (day

temperature 37 °C, >90% RH) in a growth cham-

ber (***, P < 0.001). ○, japonica-type cultivars;

●, non-japonica cultivars. Numbers with symbols

represent the following cultivars: 1, Shanguichao;

2, WAB450-1-B-P-38-HB; 3, Takanari; 4, IR72;

5, IR65564-44-2-2; 6, Banten; 7, Somewake; 8,

Homura3; 9, Koshihikari; 10, Nipponbare; 11,

Kokuryoumiyako; 12, Ginbouzu; 13, Husakushi-

razu; 14, Takenari; 15, Tairaippon; 16, Magatama;

17, Kameji2; 18, Kinmaze (MATSUI et al. 2005).

Fig. 4. Relationship between dehiscence at the basal part

of the thecae under normal conditions (in a semi-

cylindrical structure covered with cheesecloth)

and that under conditions of high temperature and

humidity (day temperature 37 ºC, >90% RH) in a

growth chamber (***, P < 0.001). Symbols and

numbers are the same as those in Fig. 3 (MATSUI

et al. 2005).


mer of 2006 in the experimental paddy field of Yangtze

University (Jingzhou City, 112º 09′ E, 30º 21′ N, elev.

32 m asl), in the western part of China’s Jianghan Ba-

sin. Twelve hybrid rice cultivars commonly cultivated

in this region were planted, with two replications. Each

plot measured 3 m (east–west) by 2 m (north–south).

Cultivation followed the currently employed local

farming methods. The pollination and seed set of flo-

rets that flowered from 14 to 16 August were examined.

Cultivars that flowered during this period were used in

the study.

The maximum temperature during the observation

period was 35.5 ºC. The length of the basal dehiscence

was significantly positively correlated with the number

sufficiently pollinated florets (Fig. 5) and with seed set

(Fig. 6) across seven hybrid rice cultivars that varied in

the length of the basal dehiscence. These correlations

demonstrate that long basal dehiscence is a major con-

tributor to stable fertilization under high temperatures

in the field through its ability to stabilize pollination.

Case 2: Nueva Ecija and Los Baños, the Philippines

The International Rice Research Institute (IRRI)

is trying to develop new breeding lines in preparation

against the high temperatures that will occur under

global warming, because few donor accessions with

heat tolerance are currently available in IRRI’s Interna-

tional Rice Genebank (IRG). By the 2008 dry season,

56 lines had been selected from about 500 accessions

based on their high seed set percentage under high tem-

peratures in the field. We examined the relationship be-

tween anther dehiscence and tolerance of high tempera-

tures for 18 of these 56 lines.

We examined the extent of both anther dehiscence

and pollination at PhilRice, the Philippine Rice Re-

search Institute, in Nueva Ecija, the Philippines. The

selected 56 lines had been planted in three 5-m rows

in a randomized block design. We used 8 of these lines

that flowered on 25 April and 10 of these lines that

flowered on 28 April in our analysis. The wind veloci-

ties at the time of flowering on both dates were about

1.5 m/s. The temperatures at flowering on 25 and 28

April were 31.4 and 30.2 ºC, respectively, and the cor-

responding daily maximum temperatures were 32.4 and

33.1 ºC, but these temperatures seemed insufficient to

induce floret sterility. We used the mean number of pol-

len grains on the stigma as an index of successful stable

pollination because almost 100% of florets had more

than 20 pollen grains on the stigma after flowering,

probably because the temperatures were insufficiently

high to decrease pollination.


that forms at the base of the thecae (15 August) and

the mean percentage of florets with more than 10

pollen grains on the stigma during the observation

period (14 to 16 August) in Jingzhou, China (*, P

≤ 0.05). Cultivars: 1, Fengliangyou No. 1; 2, IIYou

838; 3, IIYou Ming 86; 4, IIYou 084; 5, IIYou 725;

6, Shanyou 63; 7, Jingyou 63 (TIAN et al., 2010).


that formed at the basal part of the thecae and the

seed set during the observation period (14 to 16

August) in Jingzhou, China (*, P ≤ 0.05) (TIAN et

al., 2010).


Because the plants in the PhilRice study did not

exhibit high-temperature sterility during the 2008 dry

season, we used older sterility data from 2007 at IRRI

in Los Baños to provide an index of heat tolerance in

these cultivars. Materials were planted in single, unrep-

licated 5-m rows; they were then grouped according

to their flowering duration and staggered seeding was

done to ensure that flowering coincided with the hottest

time of the year (mid-April to mid-May).

The percentage of sterility closely correlated with

the number of pollen grains deposited on the stigma

(Fig. 7). This close correlation suggests that the num-

ber of pollen grains can be a useful index of pollination

stability; the main cause of sterility in this region was

poor pollination. The length of the basal dehiscence of

the anthers was significantly positively correlated with

the number of pollen grains on the stigma (Fig. 8A)

and significantly negatively correlated with the percent-

age sterility (Fig. 9A). The total of the basal and api-

cal dehiscence lengths was correlated with the number

of pollen grains (Fig. 8B) and sterility (Fig. 9B) more

closely than basal dehiscence alone, and the correlation

between basal and apical dehiscence was not signifi-

cant. Apical dehiscence was not significantly correlated

with pollen number and sterility (data not shown).

The significant correlations between the length of

the basal dehiscence and the pollen number and seed set

support our hypothesis that a long dehiscence enables

high seed set at high temperatures. However, the in-

crease in the strength of the correlation when the length

of the apical dehiscence was included suggests that the

latter parameter also plays a role in successful pollina-

tion at high temperatures. For the set of cultivars that

we examined in the Philippines, the length and range

Fig. 7. Relationship between the number of pollen grains

deposited on the stigma and percentage sterility

(IRRI 2007 data, dry season) for 18 cultivars (***,

P < 0.001). Flowering dates: ●, 25 April 2008;

○, 28 April 2008 (PhilRice; MATSUI et al., unpub-

lished).

Fig. 8. Relationships between the number of pollen grains deposited on the stigma after flowering

and (A) the length of the basal dehiscence and (B) the total length of the apical and basal de-

hiscences. Values are for all cultivars combined. Flowering dates (2008 PhilRice data): ●, 25

April; ○, 28 April. *, P < 0.05; **, P < 0.01 (Matsui et al., unpublished).


of the apical dehiscence were considerably wider than

those of the cultivars studied in the Jingzhou and growth

chamber experiments (Fig. 10). Moreover, in the Philip-

pines cultivars, the length of the apical dehiscence was

not significantly correlated with the length of the basal

dehiscence, whereas the correlation was significant for

the Jingzhou cultivars. These factors might accentuate

the role of apical dehiscence. A large apical dehiscence

may cause dispersal of many pollen grains at the start

of floret opening and may contribute to self-pollination,

although the distance from the apical dehiscence to the

stigma is somewhat farther than the distance from the

basal dehiscence.

Conclusions

Our observations have revealed that the length

of the anther dehiscence is an important factor that

increases pollination stability and fertilization under

high-temperature field conditions. The correlation be-

tween anther dehiscence and pollination was lower un-

der field conditions than in an artificially controlled en-

vironment, but the relationship was nonetheless strong

and statistically significant. Despite the uncertainties

of pollination and the many factors that have not yet

been studied, such as the effect of sudden breezes, the

strength of the correlation suggests that dehiscence

length is sufficient to explain the strong relationship be-

tween this marker and tolerance of high temperatures.

Other factors may also be important in determin-

ing the variation in HIFS among cultivars. For example,

we have not yet examined the effects of transpirational

conductance of the panicles and flowering time, which

could also affect the degree of sterility. Even though

Fig. 9. Relationship between the percentage of sterility (2007 IRRI data) and (A) the length of the basal

dehiscence (2008 Philrice data) and (B) the total length of the apical and basal dehiscences (2008

Philrice data). Values are for all cultivars combined. Flowering date (2008 PhilRice data): ●, 25

April; ○, 28 April. **, P < 0.01; ***, P < 0.001 (MATSUI et al., unpublished).

Fig. 10. Relationship between the lengths of the apical

and basal dehiscences in the four experiments.

○, Japan chamber experiment (MATSUI et al.,

2005); □, Jingzhou field experiment (TIAN et al.,

2010); ▲, Philippines field experiment (PhilRice,

25 April 2008, unpublished); △, Philippines field

experiment (MATSUI et al., unpublished; PhilRice,

28 April 2008).


the observations reported in this paper did not cover

all the factors that potentially contribute to HIFS, the

results strongly suggest that the differences in sterility

were mainly due to differences among the cultivars in

the length of anther dehiscence. The regressions in Fig-

ures 6 and 9 suggest that improving anther dehiscence

can increase seed set by at least 15 to 30% under field

conditions. A long dehiscence therefore appears to be

a useful marker for tolerance of high temperatures by

florets in the field.

Acknowledgments

We thank Reynaldo F. Diocton IV, Fresco M.

Luquias, and Marlon V. Concepcion (PhilRice) for their

assistance in the field studies. We also thank Shouta

Yamada, Junya Yanai, Kazuhei Yoshida, and Suguru

Sawagashira (Gifu University) for their assistance with

the data collection.

References

1. ANGUS, J. (1997). Rice production. A book review on

“Modeling the Impact of Climate Change on Rice Pro-

duction in Asia.” Field Crop. Res. 52: 286-287.

2. HORIE, T., MATSUI, T., NAKAGAWA, H., and OMASA, K.

(1996). Effect of elevated CO2 and global climate change

on rice yield in Japan. In Climate Change and Plants in

East Asia. Omasa, K., Kai, K., Taoda, H., Uchijima, Z.

and Yishino, M. (eds.), Springer-Verlag, Tokyo, pp. 39-

56.

3. HORIE, T., NAKAGAWA, H., NAKANO, J., HAMOTANI, K.,

and KIM, H.Y. (1995). Temperature gradient chambers

for research on global environment change. III. A system

designed for rice in Kyoto, Japan. Plant Cell Environ.

18: 1064-1069.

4. KIM, H.Y., HORIE, T., NAKAGAWA, H., and WADA, K.

(1996). Effect of elevated CO2 concentration and high

temperature on growth and yield of rice. 1. The effect on

yield and its components of Akihikari rice. Jpn. J. Crop

Sci. 65: 644-651. (In Japanese).

5. MATSUI, T. (2005). Function of long basal dehiscence of

the theca in rice (Oryza sativa L.) pollination under hot

and humid condition. Phyton 45: 401-407.

6. MATSUI, T. (2009). Floret sterility induced by high tem-

peratures at the flowering stage in rice (Oryza sativa L.).

Jpn. J. Crop Sci. 78: 303-311. (In Japanese).

7. MATSUI, T., and KAGATA, H. (2003). Characteristics of

floral organs related to reliable self-pollination in rice

(Oryza sativa L.). Ann. Bot. 91: 473-477.

8. MATSUI, T., KOBAYASI, K., KAGATA, H., and HORIE,

T. (2005). Correlation between viability of pollina-

tion and length of basal dehiscence of the theca in rice

under a hot-and-humid condition. Plant Prod. Sci. 8:

109-114.

9. MATSUI, T., KOBAYASI, K., YOSHIMOTO, M., and

HASEGAWA, T. (2007). Stability of rice pollination in the

field under hot and dry conditions in the Riverina region

of New South Wales, Australia. Plant Prod. Sci. 10: 57-

63.

10. MATSUI, T., NAMUCO, O.S., ZISKA, L.H., and HORIE, T.

(1997). Effects of high temperature and CO2 concentra-

tion on spikelet sterility in indica rice. Field Crops Res.

51: 213-219.

11. MATSUI, T., OMASA, K., and HORIE, T. (1999). Mecha-

nism of anther dehiscence in rice (Oryza sativa L.). Ann.

Bot. 84: 501-506.

12. MATSUI, T., OMASA, K., and HORIE, T. (2000). High tem-

perature at flowering inhibits swelling of pollen grains, a

driving force for thecae dehiscence in rice (Oryza sativa

L.). Plant Prod. Sci. 3: 430-434.

13. MATSUI, T., OMASA, K., and HORIE, T. (2001). The dif-

ference in sterility due to high temperature during the

flowering period among japonica-rice varieties. Plant

Prod. Sci. 4: 90-93.

14. NAKAGAWA, H., HORIE, T., and MATSUI, T. (2003). Ef-

fects of climate change on rice production and adaptive

technologies. In Rice Science: Innovations and Impact

for Livelihood. Mew, T.W., Brar, D.S., Peng, S., Dawe,

D. and Hardy, B. (eds.), International Rice Research In-

stitute, Los Baños, pp. 635-658.

15. OSADA, A., SACIPLAPA, V., RAHONG, M., DHAMMANU-

VONG, S., and CHAKRABANDHO, H. (1973). Abnormal

occurrence of empty grains of indica rice plants in the

dry, hot season in Thailand. Proc. Crop Sci. Soc. Jpn. 42:

103-109.

16. SATAKE, T., and YOSHIDA, S. (1978). High temperature-

induced sterility in indica rice at flowering. Jpn. J. Crop

Sci. 47: 6-10.

17. TIAN, X., MATSUI, T., Li, S., YOSHIMOTO, M., KOBAYASI,

K., and HASEGAWA, T. (2010). Heat-induced floret steril-

ity of hybrid rice (Oryza sativa L.) cultivars under humid

and low wind conditions in the field of Jianghan Basin,

China. Plant Prod. Sci. 13: 243-251.


質疑応答司会：松井先生，どうもありがとうございました。地球の温暖化に向けて，葯の裂開ですかね。葯の裂開の大きいものの方が，高温に対する耐性が強いという，そういうことで，なおかつ遺伝的な変異もあるということになりますと，いわゆるあの，育種が可能だなということになります。とても素晴らしい講演だったと思いますが，どなたかご質問，ご意見がございましたら，どうぞ。西尾先生どうぞ。西尾：東北大学の西尾でございます。高温障害，高温不稔でその日本の特にあきたこまちなんかの方が，インディカ米よりも強いというのは，大変興味深く聞かしていただきましたですけれども。で，この，たくさんの品種を調べられた図もありまして，その中で金南風なんかが非常に強そうな評価になっていますが，ほかにもっといろいろ調べて非常に強いというふうなものは見つけておられますか。松井：非常に強いっていうものは見つけてないですね。昔，1978年に佐竹さん，吉田さんの ₂人がフィリピンの国際稲研究所で，耐性の研究をされて，そのときに N22という強い品種があるということ，また，受粉が非常に安定しておるということを見つけられたのですけども，わたしの知る限りでは，N22はあきたこまちより，少し強いぐらいですが，N22を超えるような強さの品種というのは，わたしは見たことがないですね。その後，国際稲研究所でもいろいろスクーリングをやっていますが，結局受粉の安定性という視点からみますと，N22は ₁番強いと感じますね。もちろん，その受粉だけで耐性が決まるわけではないですから，N22よりもわずかに稔実が高温条件で高い品種もあるみた

いですけれども，受粉という面から見ると，それほど強い品種はない。40度を超えるとやっぱり稔実率は極端にどの品種も低下してくる，ということです。司会：よろしいですか。ほかにございませんでしょうか。ちょっとわたしの方からお願いしたいと思いますが。高温を考える場合に，湿度との関係もちろんあると思うのですが，どのくらいの，まあこれは例えば日本の品種だとちょっと強そうですので，日本の場合はもうちょっと高くまでいけるのかなと思いますが，一般的に何度ぐらいが，高温障害が起こる温度だというふうに考えたらいいのでしょうか。松井：そうですね。この問題には湿度と風の条件がいろいろ絡みますが，大体体温がやはり 35

度を超えると不稔が出始めるということですね。ですから結構思ったよりも低い。中部地方，わたしが今住んでいるところは，結構暑い。関西も暑いですけど。最高気温で言うと 35度を超える日が結構あります。それはまあ，高温不稔がぎりぎり起こらないぐらいのことかもしれないですね。2007年に農環研の方と作物研の方が，関東と中部地域でかなり高温が出たということで，調査されましたが，そういう暑い年によくよく見てみると，場合によっては，田んぼによってはですね，10パーセントとか，10

数パーセントの不稔が出始めているということですね。これがもう少し高くなると，急激に出てくるのかどうかというのは，分からないですけども。でも，もう，大抵，その大げさに言っているのかもしれない。分からないのですけども，ファイトトロンの中で実験すると，不稔が出だすと，まあ， ₁～ ₂度上がれば，すぐに稔実率が 50パーセントから 30パーセントにも

イネの高温不稔の発生と耐性のメカニズム

松　井　　　勤

岐阜大学


なるものですから，さらに（気温が）上がるとひょっとしたら（稔実率や収量が）減るのではないかと考えております。司会：ほかにございませんでしょうか。あの最もクリティカルな時期っていうのは，やはり花粉が飛散するとき，開花のときというふうに考えていいですか。松井：それは，実はわたし，昔からの研究で，そういうことになっているのですけども，最近，そうでもないかもしれないなと。その高温の影響の積算的な影響っていうのですかね，そういうものはないっていうふうに言われてきたのですが，よくよく見ていると，前日が暑くて，開花の当日も暑い場合には，不稔はかなり発生しやすいなというふうに感じています。それちょっと数字をはっきりとったことはないですけども。やはり連続した高温には弱いのではないかなと感じます。それからオーストラリアなんかですと，開花前日の高温でむしろ不稔が発生します。ああいう極端な乾燥地域っていうのですかね，暑くなるところというのは，午後にぐいぐい気温が上がっていって，日本なんかは大体２時ぐらいで終わりですが， ₃時４時 ₅時とこう気温が上がっていったりするものですから。そういうふうな地域では，開花時刻の気温は低くても，その開花以前の日のダメージって

いうのが，開花のときに出てきてですね，不稔が発生するということがあるようですね。司会：どうぞ。なにか。中川：生物研の中川ですけども，今の質問と関連して，よく花粉の稔性をやるときに，多分，インゲンか何かで夜温について議論され，イネの場合には夜温が影響与えたという，そういう例は何か・・・松井：ファイトトロンの実験で実験をすると，大体 30度を超えると，夜温の影響も出てきます。夜温が高いことによって，花粉の発芽（不良）を通じてですね，不稔の発生があるということなのですけども。屋外ではちょっとなかなか30度，今のところ 30度というのはあまりない。暑いところは，結構夜涼しいということがあります。　　いつの温度が影響あるのかを特に屋外では切り離して，調査することは難しいので。ファイトトロンの実験では，30度が（受精に影響を与えない限界の温度）ということが言われています。司会：時間になりましたが，ほかにもしございましたら。ないよう，ございませんでしょうか。そしたら松井先生，どうもありがとうございました。

（拍手）

53

Introduction

Dormancy is a phenomenon that causes a temporal

arrest of growth and development of the plant or plant

tissues such as bud and seed. This mechanism is an im-

portant strategy to overcome environmental stresses by

preventing plant growth under adverse environmental

conditions, such as heat, cold, and drought, while al-

lowing plant growth to resume under favorable condi-

tions. Dormancy provides both a benefit and a cost to

plants, especially cultivated crops, since weaker dor-

mancy causes entrained germination and higher yield

but carries the potential risk of pre-harvest sprouting;

conversely, deeper dormancy causes lower germina-

bility but prevents pre-harvest sprouting. Pre-harvest

sprouting often occurs if the appropriate temperature

and humidity are present at seed maturity, resulting in

a reduction of grain quality. Hence, it is very impor-

tant in breeding programs of rice as well as other cereal

crop species to select for seed dormancy at a level ap-

propriate for the environmental conditions the seed will

encounter.

(HILHORST 2007) has proposed a classification of

dormancy based on timing. In this system, dormancy

is divided into two classes: primary dormancy, which

is established during seed development, and second-

ary dormancy, which is induced in the mature seed by

inadequate conditions for germination. Furthermore,

primary dormancy is divided into two subcategories,

embryonic dormancy, which is due to the regulation of

growth by the embryo itself, and mechanical or seed-

coat-imposed dormancy, which is physical suppression

of embryonic growth. Embryonic growth potential is

regulated mainly by the balance of the phytohormones

abscisic acid (ABA) and gibberellins (GA). ABA plays a

crucial role in dormancy induction and maintenance and

in seed maturation, and produces desiccation tolerance

in the seed (GUBLER et al. 2005). Genetic approaches

have been used to understand dormancy in monocots.

For example, two maize viviparous mutants that led to

precocious germination, viviparous 1 (vp1) (McCARTY

et al. 1991) and vp14 (TAN et al. 1997), were found to

have reduced ABA sensitivity and reduced ABA syn-

thesis, respectively. During seed development and mat-

uration, ABA synthesis and ABA sensitivity are equally

important. The acquired seed dormancy is released by

after-ripening, resulting in non-dormant seed, and the

seed is then able to germinate under suitable humidity

and temperature condition.

In cereals such as rice, wheat, and barley, recent

progress in genome analysis has allowed scientists to

detect quantitative trait loci (QTLs) involved in seed

dormancy or pre-harvest sprouting. In particular, many

QTLs have been identified in rice. In spite of such

progress, the genetic control of seed dormancy is still

unclear because of the lack of information on gene

structures and functions. In this review, we summarize

current information on genetically identified rice QTLs

and some examples of molecular cloning of seed dor-

mancy QTLs. Furthermore, based on the chromosomal

locations of the QTLs that have been detected, we dis-

cuss possible relationships between reported rice QTLs

and rice orthologs of Arabidopsis genes related to seed

dormancy. Finally, we discuss current strategies for the


Genetic Control of Seed Dormancy in Rice

Kazuhiko SUGIMOTO1*, Salem MARZOUGI2 and Masahiro YANO1,2

1 National Institute of Agrobiological Science,

2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan2 Graduate School of Life and Environmental Sciences, University of Tsukuba,

1-1-1 Ten-noudai, Tsukuba, Ibaraki 305-8572, Japan

54 Kazuhiko SUGIMOTO, Salem MARZOUGI and Masahiro YANO

genetic and molecular analysis of seed dormancy in

rice.

Rice QTLs related to seed dormancy and pre-har-

vest sprouting

During the last decade, many QTLs for seed dor-

mancy in rice have been mapped using molecular mark-

ers. The chromosomal locations of these QTLs are sum-

marized in Fig. 1. Genetic dissection of seed dormancy

using isozyme markers has revealed QTLs on chromo-

somes 3, 6 (two loci), 7, and 12 (WAN et al. 1997).

By using RFLP markers, five QTLs were detected in

backcross inbred lines (BILs) derived from crosses be-

tween a japonica cultivar, Nipponbare, and an indica

cultivar, Kasalath; these were located on chromosomes

3, 5, 7 (two loci), and 8 (LIN et al. 1998; TAKEUCHI et

al. 2003). It should be noted that four different QTLs

for seed dormancy were detected by testing the same

BILs under greenhouse conditions (Miura et al. 2002).

These results suggested that the level of seed dorman-

cy is significantly affected by environmental factors

such as temperature during seed maturation (IKEHASHI

1972). Many QTLs for dormancy have been detected

in the progeny of crosses between japonica and indica

cultivars (DONG et al. 2003; GAO et al. 2008; GUO et al.

2004; WAN et al. 2005; YOU et al. 2006). QTLs have

also been detected in populations derived from weedy

rice, on chromosome 1, 4, 6, 7 (two loci), 8, and 12

(GU et al. 2004; GU et al. 2005). Many dormancy-re-

lated QTLs have been detected in crosses with wild rice

(O. rufipogon) (CAI and MORISHIMA 2002; LEE et al.

2005; THOMSON et al. 2003). It is noteworthy that the

chromosomal locations of the QTLs summarized in

Fig. 1 were concentrated in a limited number of regions

(Fig. 1). Although it is unknown whether those QTLs

mapped in the same chromosomal region, this implies

that a relatively small number of genetic factors might

be involved in natural variation in seed dormancy.

The gene qLTG3-1 was first reported as a gene

regulating germinability under low temperatures (FU-

JINO et al. 2008). It regulates weakening of the epiblast;

thus, its function is controlling mechanical resistance to

coleoptile growth. It was also reported that pre-harvest

sprouting resistance of Koshihikari, a leading japoni-

ca cultivar, is probably due to the loss of function of

qLTG3-1 (HORI et al. 2010). Therefore, it is possible to

think that qLTG3-1 is also involved in seed dormancy,

especially mechanical dormancy. The candidate region

of qSD12 has been delimited to less than 75 kb, and

PIL5 and bHLH related to Arabidopsis seed germina-

tion regulators were found (GU et al. 2009). The plants

carrying a qSD12 allele from SS18-2 exhibited an in-

creased (2-fold) level of ABA in the endosperm at 10

days after anthesis compared with plants carrying only

the allele from the cultivated parent EM93-1, suggest-

ing that qSD12 may regulate ABA content at the seed

dormancy acquisition stage.

Recently, Seed dormancy 4 (Sdr4), a QTL on the

long arm of chromosome 7, was cloned (SUGIMOTO et

al. 2010). Based on its predicted amino acid sequence,

Sdr4 encodes a novel protein that showed no homology

with known proteins. Sdr4 expression was upregulated

by seed maturation regulator OsVP1 and could nega-

tively regulate germination-related genes. In an sdr4

mutant, dormancy was lost but ABA responsiveness of

the LEA genes was retained, suggesting that Sdr4 may

be a seed dormancy–specific regulator. Furthermore,

Sdr4 was expressed in seed, especially in the embryo,

and the size of the sdr4 mutant embryo was 60% larger

than that of wild type. These results suggested that the

sdr4 mutant had reduced ability to arrest the growth of

the embryo and induce dormancy.

Rice orthologs of Arabidopsis seed dormancy related

genes located near the QTLs

In Arabidopsis, many dormancy-related genes

have been isolated by using mutants (Table 1) (Hilhorst

2007). Therefore, an interesting question is whether

rice orthologs of these Arabidopsis genes are involved

in natural variation in seed dormancy. We have summa-

rized co-localization of these orthologs and previously

mapped rice QTLs (Fig. 1). Several rice orthologs of

Arabidopsis seed dormancy genes were found to be co-

localized with rice QTLs on chromosomes 1S, 1L, 4L,

55GENETIC CONTROL OF SEED DORMANCY IN RICE

Table 1 Rice orthologs of Arabidopsis genes related to ABA signaling, embryogenesis, and dormancy/germination.

Orthologs were selected based on the SALAD database (http://salad.dna.affrc.go.jp/salad/en/). In the case of rice

orthologs that did not already have a name, we tentatively named these with the Arabidopsis gene name followed by

an “L” (column 3). If no ortholog could be selected, the gene was indicated as “unclear” in column 4. The positions

listed in column 5 are based on IRGSP genome sequence Build 4.

At gene AGI Os gene Orthologue Position Full-lengthABI1 At4g26080 ABI1L Os05g0592800 chr5:29620026..29623599ABI2 At5g57050 ABI2L Os05g0537400 chr5:26766712..26769064ABI5 At2g36270 TRAB1 Os08g0472000 chr8:23322479..23328849AIP2 At5g20910 AIP2L Os09g0434200 chr9:16598274..16601628 AK103447

AMP1 At3g54720 OsAMP1 Os03g0789900 chr03:33731156..33732990ERA1 At5g40280

ERA1LOs01g0737800 chr1:32530986..32535085 AK073299

RHA2a At1g15100 unclearPYR1 At4g17870 PYR1L Os10g0573400 chr10:23221607..23222695 AK061525GTG1 At1g64990

GTG1L Os04g0600800 chr4:30710067..30714823GTG2 At4g27630GPA1 At2g26300 GPA1L Os05g0333200 chr5:15587303..15610837GUN5 At5g13630 GUN5L Os03g0323200 chr3:11762832..11770250 AK067323

AtPirin1 At3g59220Pirin1L1 Os03g0845000 chr3:36374983..36376946 AK105971Pirin1L2 Os09g0484800 chr9:19361595..19364026 AK109548

ABH1 At2g13540 ABH1L Os03g0347200 chr3:13028130..13040179 AY017415SAD1 At5g48870 SAD1L Os05g0389300 chr5:18845158..18847627HYL1 At1g09700 unclear

AtNCED4 At4g19170OsNCED1 Os02g0704000 chr02:29882096..29884173 AK064824OsNCED2 Os12g0435200 chr12:14389427..14391376 AK120176

AtNCED3/9At3g14440

At1g78390

OsNCED3 Os03g0645900 chr03:25770177..25772236 AY838899

OsNCED4 Os07g0154100 chr07:2902838..2904981 AK119780

CYP707A1 At4g19230OsABA8ox1 Os02g0703600 chr2:29882578..29885370 AK067007CYP707A2 At2g29090

CYP707A3 At5g45340

CYP707A4 At3g19270OsABA8ox2 Os08g0472800 chr8:23376619..23382168 AK120757OsABA8ox3 Os09g0457100 chr9:17905988..17909042

ABI3 At3g24650 OsVP1 Os01g0911700 chr1:41479018..41482835 AK073805LEC2 At1g28300 LEC2L Os08g0101000 chr8:62575..66639 AK107456FUS3 At3g26790 OsLFL1 Os01g0713600 chr1:31393920..31398707 AK109920VAL1 At4g21550

VAL1L Os07g0679700 chr7:29446630..29453161 AK101356VAL2 At2g30470VAL3 At4g32010LEC1 At1g21970 LEC1L Os06g0285200 chr6:10136824..10137874 AY062183

DOG1 At5g45830DOG1L1 Os05g0560200 chr5:27965135..27966137DOG1L2 Os01g0159000 chr1:3090359..3091698 AK106273

HUB1(RDO4) At2g44950 OsBre1A Os10g0565600 chr10:22843975..22851110 AK103864HUB2 At1g55250 OsBre1B Os04g0550400 chr4:27949863..27958575 AK067475

MARD1 At3g63210MARD1L1 Os10g0422600 chr10:15417794..15420086 AK109893MARD1L2 Os03g0665200 chr3:26912605..26915883 AK066202

DAG1 At3g61850DAG1L Os02g0673700 chr2:28322580..28325215 AB028130

DAG2 At2g46590DEP1 At1g70910 unclear

ATHB20 At3g01220 ATHB20L Os10g0404900 chr10:14162346..14163619ABI4 At2g40220 ABI4L Os05g0351200 chr5:16578817..16579626


5L, 8L, and 8S.

Among the orthologs mapped to chromosome 1L,

OsLFL1 encodes a type of B3 domain transcription fac-

tor that resembles the Arabidopsis regulators of seed

development and dormancy, FUS3. An ERA1-like gene

was also located near the QTLs on chromosome 1L.

OsLFL1 was reported to be a regulator of flowering

time in rice, and ERA1 is involved in the ABA signal

transduction pathway in Arabidopsis (CUTLER et al.

1996; PENG et al. 2008).

Fig. 1. Positions of rice QTLs and rice orthologs of Arabidopsis genes involved in seed dormancy and germination regula-

tion

Locations of QTLs and orthologs were determined based on the International Rice Genome Sequencing Project

(IRGSP) rice genome sequence Build 4. Rice orthologs of Arabidopsis genes are indicated to the left of each chro-

mosome (gray bar): genes related to ABA signaling are indicated in brown, genes related to embryogenesis are in

green, and dormancy and germination-specific genes are in sky blue. QTLs related to seed dormancy and germination

are indicated to the right of each chromosome: colors indicate the source of the information (see key). Vertical bars

show the position of each QTL as defined by flanking markers; arrowheads within the bars point to the position of the

nearest marker. QTLs for which flanking markers have not been reported are drawn as ovals. QTLs without detailed

position information were not included on the map”? If so, please make this change. In the figure key, reports in which

QTLs were detected in progenies of japonica × indica and cultivar × wild rice crosses are indicated by open and

closed circles, respectively. The QTLs reported by GU et al. were derived from crosses of cultivated × weedy rice.


TRAB1 and OsABA8ox2 were located near QTLs

mapped to chromosome 8L. TRAB1 was reported to

be an ABRE gene regulator that is regulated by phos-

phorylation in the presence of ABA (Hobo et al. 1999).

OsABA8ox deactivates ABA by converting it to pha-

seic acid (PA), thus leading to germination (ZHU et al.

2009). Lec2L, a rice gene similar to LEC2 from Arabi-

dopsis, also encodes a B3 domain transcription factor

that regulates seed morphogenesis and seed dormancy;

Lec2L is located on chromosome 8S. Based on the pre-

dicted functions from Arabidopsis gene analysis, these

genes may affect seed development or ABA response.

In general, it is unlikely that genes related to ABA

synthesis and signal transduction would be involved

in natural variation because they would be expected to

have a detrimental effect on plant fitness. Therefore,

study of such genes should focus on those genes that

appear to be specific to dormancy. These types of stud-

ies have been done with the naturally occurring Arabi-

dopsis ecotype Cape Verde Islands (Cvi), which shows

strong dormancy (BENTSINK et al. 2006; BENTSINK et

al. 2003). QTL analysis of recombinant inbred lines de-

rived from a cross between Landsberg erecta (Ler) and

Cvi revealed 14 QTLs related to seed dormancy. One

of the QTLs with the largest effects explained 12% of

the variance in seed dormancy; from this QTL, DELAY

OF GERMINATION 1 (DOG1) was isolated. DOG1

encodes a novel unknown-function protein that is re-

lated to DNA-binding proteins but differs from known

proteins involved in ABA/GA signal transduction or

biosynthesis. We have identified two rice orthologs of

DOG1, DOG1L1 and DOG1L2, on chromosomes 5L

and 1S, respectively. The novel characteristics of Ara-

bidopsis DOG1 suggest that these orthologs should be

the focus of future study.

The Arabidopsis gene HISTONE MONOUBIQUI-

TINATION 1 (HUB1) was isolated based on a mutant

phenotype of reduced of seed dormancy; that study re-

vealed that seed dormancy is also regulated by chroma-

tin modification (LIU et al. 2007). In the hub1 mutant,

the expression level of DOG1 was reduced, suggesting

that HUB1 positively regulates DOG1 expression. The

rice HUB1- and HUB2-like genes (HUB1L and HUB2L,

located on chromosomes 10L and 4L, respectively) is

also one of the candidate genes.

Conclusion

Although many rice QTLs for seed dormancy have

been detected, the molecular basis of seed dormancy in

rice remains to be clarified. Knowledge from Arabidop-

sis studies is expected to deepen our understanding of

seed dormancy in rice. In some cases, rice orthologs of

Arabidopsis seed dormancy genes have been co-local-

ized with rice QTLs, but the relationships between these

orthologs and the nearby QTLs still need to be verified.

Even if we demonstrate the involvement of these genes

with seed dormancy in rice, however, that information

might not be useful for improvement of rice pre-harvest

sprouting. We need to focus on those genes specific to

seed dormancy, because genes functioning in the up-

stream part of the ABA signal transduction pathway

might have pleiotropic effects on other plant charac-

teristics. The cloning of rice Sdr4 clearly demonstrated

that sequence variation in such a gene can generate phe-

notypic variation in seed dormancy without any other

pleiotropic effects, suggesting that such genes would

be good candidates for the improvement of pre-harvest

sprouting. Cloning of other QTLs involving in natural

variation for seed dormancy will give us a unique op-

portunity to improve rice cultivars as well as to deepen

our understanding of seed dormancy.

References

1. BENTSINK, L., JOWETT, J., HANHART, C.J. and KOORN-

NEEF, M. (2006) Cloning of DOG1, a quantitative trait

locus controlling seed dormancy in Arabidopsis. Pro-

ceedings of the National Academy of Sciences of the

United States of America 103: 17042-17047.

2. BENTSINK, L., YUAN, K., KOORNNEEF, M. and VREUG-

DENHIL, D. (2003) The genetics of phytate and phos-

phate accumulation in seeds and leaves of Arabidopsis

thaliana, using natural variation. TAG. Theoretical and

applied genetics 106: 1234-1243.

3. CAI, W. and MORISHIMA, H. (2002) QTL clusters reflect

character associations in wild and cultivated rice. TAG.

Theoretical and applied genetics 104: 1217-1228.


4. CUTLER, S., GHASSEMIAN, M., BONETTA, D., COONEY, S.

and McCOURT, P. (1996) A protein farnesyl transferase

involved in abscisic acid signal transduction in Arabi-

dopsis. Science New York, N.Y 273: 1239-1241.

5. DONG, Y., TSUZUKI, E., KAMIUNTEN, H., TERAO, H., LIN,

D., MATSUO, M. and ZHENG, Y. (2003) Identification of

quantitative trait loci associated with pre-harvest sprout-

ing resistance in rice (Oryza satiba L.). Field Crops Res.

81: 133-139.

6. FUJINO, K., SEKIGUCHI, H., MATSUDA, Y., SUGIMOTO, K.,

ONO, K. and YANO, M. (2008) Molecular identification

of a major quantitative trait locus, qLTG3-1, controlling

low-temperature germinability in rice. Proceedings of

the National Academy of Sciences of the United States of

America 105: 12623-12628.

7. GAO, F.Y., REN, G.J., LU, X.J., SUN, S.X., LI, H.J., GAO,

Y.M., LUO, H., YAN, W.G. and XHANG, Y.Z. (2008) QTL

analysis for resistance to preharvest sprouting in rice

(Oryza sativa). Plant Breed. 127: 268-273.

8. GU, X.Y., KIANIAN, S.F. and FOLEY, M.E. (2004) Mul-

tiple loci and epistases control genetic variation for seed

dormancy in weedy rice (Oryza sativa). Genetics 166:

1503-1516.

9. GU, X.Y., KIANIAN, S.F., HARELAND, G.A., HOFFER, B.L.

and FOLEY, M.E. (2005) Genetic analysis of adaptive

syndromes interrelated with seed dormancy in weedy

rice (Oryza sativa). TAG. Theoretical and applied genet-

ics 110: 1108-1118.

10. GU, X.Y., LIU, T., FENG, J., SUTTLE, J.C. and GIBBONS,

J. (2009) The qSD12 underlying gene promotes abscisic

acid accumulation in early developing seeds to induce

primary dormancy in rice. Plant molecular biology.

11. GUBLER, F., MILLAR, A.A. and JACOBSEN, J.V. (2005)

Dormancy release, ABA and pre-harvest sprouting. Cur-

rent opinion in plant biology 8: 183-187.

12. GUO, L., ZHU, L., XU, Y., ZENG, D., WU, P. and QIAN,

Q. (2004) QTL analysis of seed dormancy in rice (Oryza

sativa L.). Euphytica 140: 155-162.

13. HILHORSt, H.W.M. (2007) in Seed Development, Dor-

mancy and Germination. eds Bradford KJ, Nonogaki H

(Blackwell Publishing, Sheffield UK), pp. . Blackwell,

Oxford, UK.

14. HOBO, T., KOWYAMA, Y. and HATTORI, T. (1999) A bZIP

factor, TRAB1, interacts with VP1 and mediates abscisic

acid-induced transcription. Proceedings of the National

Academy of Sciences of the United States of America 96:

15348-15353.

15. HORI, K., SUGIMOTO, K., NONOUE, Y., ONO, N., MAT-

SUBARA, K., YAMANOUCHI, U., ABE, A., TAKEUCHI, Y.

and YANO, M. (2010) Detection of quantitative trait loci

controlling pre-harvest sprouting resistance by using

backcrossed populations of japonica rice cultivars. TAG.

Theoretical and applied genetics in press.

16. IKEHASHI, H. (1972) induction and test of dormancy of

rice seeds by temperature condition during maturation.

Jpn J Breed. 22: 209-216.

17. LEE, S.J., Oh, C.S., SUH, J.P., McCOUCH, S.R. and AHN,

S.N. (2005) Identification of QTLs for domestication-

related and agronomic traits in an Oryza sativa x O. rufi-

pogon BC1F7 population. Plant Breed. 124: 209-219.

18. LIN, S.Y., SASAKI, T. and YANO, M. (1998) Mapping

quantitative trait loci controlling seed dormancy and

heading date in rice, Oryza sativa L., using backcross

inbred lines. TAG. Theoretical and applied genetics 96:

997-1003.

19. LIU, Y., KOORNNEEF, M. and SOPPE, W.J. (2007) The ab-

sence of histone H2B monoubiquitination in the Arabi-

dopsis hub1 (rdo4) mutant reveals a role for chromatin

remodeling in seed dormancy. The Plant cell 19: 433-

444.

20. McCARTY, D.R., HATTORI, T., CARSON, C.B., VASIL,

V., LAZAR, M. and VASIL, I.K. (1991) The Viviparous-1

developmental gene of maize encodes a novel transcrip-

tional activator. Cell 66: 895-905.

21. MIURA, K., LIN, Y., YANO, M. and Nagamine, T. (2002)

Mapping quantitative trait loci controlling seed longev-

ity in rice ( Oryza sativa L.). TAG. Theoretical and ap-

plied genetics 104: 981-986.

22. PENG, L.T., SHI, Z.Y., LI, L., SHEN, G.Z. and ZHANG, J.L.

(2008) Overexpression of transcription factor OsLFL1

delays flowering time in Oryza sativa. Journal of plant

physiology 165: 876-885.

23. SUGIMOTO, K., TAKEUCHI, Y., EBANA, K., MIYAO, A., HI-

ROCHIKA, H., HARA, N., ISHIYAMA, K., KOBAYASHI, M.,

BAN, Y., HATTORI, T. and YANO, M. (2010) Molecular

cloning of Sdr4, a regulator involved in seed dormancy

and domestication of rice. Proceedings of the National

Academy of Sciences of the United States of America

107: 5792-5797.

24. TAKEUCHI, Y., LIN, S.Y., SASAKI, T. and YANO, M.

(2003) Fine linkage mapping enables dissection of

closely linked quantitative trait loci for seed dormancy

and heading in rice. TAG. Theoretical and applied genet-

ics 107: 1174-1180.

25. TAN, B.C., SCHWARTZ, S.H., ZEEVAART, J.A. and Mc-

CARTY, D.R. (1997) Genetic control of abscisic acid

biosynthesis in maize. Proceedings of the National

Academy of Sciences of the United States of America 94:


12235-12240.

26. THOMSON, M.J., TAI, T.H., McCLUNG, A.M., LAI, X.H.,

HINGA, M.E., LOBOS, K.B., XU, Y., MARTINEZ, C.P. and

McCOUCH, S.R. (2003) Mapping quantitative trait loci

for yield, yield components and morphological traits in

an advanced backcross population between Oryza ru-

fipogon and the Oryza sativa cultivar Jefferson. TAG.

Theoretical and applied genetics 107: 479-493.

27. WAN, J., MAKAZAKI, K., KAWAURA, K. and IKEHASHI, H.

(1997) Identification of marker loci for seed dormancy

in rice (Oriza sativa L.). Crop Sci. 37: 1759-1763.

28. WAN, J.M., CAO, Y.J., WANG, C.M. and IKEHASHI, H.

(2005) Quantitative trait loci associated with seed dor-

mancy in rice. Crop science 45: 712-716.

29. YOU, J., LI, Q., YUE, B., XUE, W.Y., LUO, L.J. and XIONG,

L.Z. (2006) Identification of quantitative trait loci for

ABA sensitivity at seed germination and seedling stages

in rice. Acta Genet Sinica 33: 532-541.

30. ZHU, G., YE, N. and ZHANG, J. (2009) Glucose-induced

delay of seed germination in rice is mediated by the sup-

pression of ABA catabolism rather than an enhancement

of ABA biosynthesis. Plant & cell physiology 50: 644-

651.


質疑応答司会：ありがとうございました。講演の中でもあったと思うんですけれども，すでにトウモロコシですとか，シロイヌナズナで先行する研究あるわけですけれども，そういった中で，イネの QTLの解析から，これまで見つかってないような遺伝子機能が明らかになったということを，ご紹介したかったと思いますけれども。ただ今のご講演に対しまして，ご質問，ご意見等ございましたら，よろしくお願いいたします。どうぞ。まず，所属と名前お願いします。久保山：茨城大学の久保山です。マイクロアレイで Sdr ４のミュータントと比較されていましたけど，ニアアイソジェニックラインとかとの比較っていうようなことはされてないんですか。もし穂発芽っていうことに着目して，農業形質的なことに着目する，そちらの比較の方が面白いと思うんですけれど。杉本：はい。それは確かにおっしゃる通りだと思います。マイクロアレイ解析なかなかお金もかかりますので，ちょっと優先順位を落としましたが，実際何がこの農業形質を決めているのかということを決めるためには，主役を見つけ出すためには，それが非常に重要ではないかと，今後取り組みたいと考えています。久保山：ありがとうございます。司会：と，もしあればもう。と，じゃあ，長戸先生の方が早かったと思うんで，長戸先生。あ，いいですか。長戸先生。長戸： ₁ つお聞きしたいのは，OsVP ₁ は休眠が始まる以前のごく初期から発現していましたが，胚の表現型は何か変わったのですか。杉本：実は切片を作って見ていけばよかったのですが，残念ながらまだ 14日目以降しか見てい

ないので，その前のどんどん丸い形にしていく段階は観察していません。今年の夏，それをぜひ観察したいと。長戸：そのミューテーションはヌルだと思います。それっていうのも，かなり。杉本：かなり落ちてるんですけども，長戸：弱い。杉本：ヌルではないかもしれないです。また，完全なヌルはまたもっと違うフェノタイプが現れる可能性はまだ残っています。長戸：Sdr ４は，何日目から発芽，発現しますか。杉本：今回は示しませんでしたが，リアルタイムで見る限り，10日でもそれなりのレベルが実際，胚では出ており，オールシードでは，あまりはっきり見えないんですが，胚のレベルで見ていますと，もう 10日目の段階でかなり出ています。 ₂ 度のピークがありまして，10日目でかなり隆起があって，後半でまあ蓄積するというパターンを示していますので，思ったより早い時期から出ていることは想定しています。長戸：発現場所は全体。杉本：全体ですが，やはり胚が濃いように見えます。長戸：根っこも。杉本：根は出ていないです。ただ根も詳細に見ているわけではありませんので。後，障害で少し動く。傷がついたところで少し遺伝子が動いています。ですが，植物体ではまだ詳しく調べてないというのもありますが，今のところ出ていません。長戸：はい。ありがとうございます。司会：まだご質問があるかと思うんですけども，時間ですので，これで終わりたいと思います。どうもありがとうございました。

（拍手）

イネ穂発芽耐性ＱＴＬの単離と種子休眠機構の解析

杉　本　和　彦

農業生物資源研究所

61

Introduction

Acetylcholine (ACh) is a well-known neurotrans-

mitter that propagates electrical stimuli across the syn-

aptic junction in animals although ACh also has been

identified in some non-neuronal tissues in animals,

plants, fungi, and bacteria (KAWASHIMA et al. 2007).

Both ACh and acetylcholinesterase (AChE) activity

have been widely reported in plants (MOMONOKI and

MOMONOKI 1991; ROSHCHINA 2001; HORIUCHI et al.

2003; WIŚNIEWSKA and TRETYN 2003; YAMAMOTO

et al. 2009).We has proposed a model for an ACh-me-

diated system in plants that results in an asymmetric

distribution of hormones and substrates in response to

gravity, as well as changes in ACh content, AChE ac-

tivity, and Ca2+ level in response to heat (MOMONOKI

1997). Indeed, after the stimulus, the AChE activity was

distributed asymmetrically at the interface between the

stele and the cortex of the gravistimulated maize seed-

lings (MOMONOKI, 1992). On the other hand, maize

seedlings that were treated first with specific AChE

inhibitor, neostigmine bromide did not respond to the

gravistimulus (MOMONOKI, 1997). The function of the

plant ACh-mediated system is still unclear, however,

despite more than 30 years of research.

Recently, our group purified maize AChE and

cloned the gene encoding it from maize seedlings. This

was the first direct evidence of plant genes and proteins

related to the ACh-mediated system (SAGANE et al.

2005). Next, the AChE protein was purified and cloned

from seedlings of the legume siratro (Macroptilium at-

ropurpureum Urb.), which we consider to be represen-

tative of the AChEs of dicots (YAMAMOTO et al. 2008).

AChE homologs are widely distributed in plants, but

the primary structure of maize AChE shows extremely

low homology with those of animal AChEs. Thus, we

presume that the different primary structures of AChEs

in monocots and dicots affect their molecular and enzy-

matic properties.

To better understand ACh-mediated systems in

plants, in this study: (1) the molecular and enzymatic

properties of purified siratro AChE were compared with

those of maize AChE and electric eel AChE, which are

representative of monocot and animal AChE, respec-

tively; (2) the maize AChE gene was overexpressed in

rice plants, and the subcellular localization of maize

AChE in transgenic rice plants was determined by im-

munohistochemistry.

The molecular and enzymatic properties of AChE

from siratro

Siratro AChE was purified using a column chro-

matography system, and the molecular and enzymatic

properties of purified siratro AChE were compared with

previously purified maize AChE and electric eel AChE.

Siratro AChE is a disulfide-linked 125-kDa homotrimer

consisting of 41- to 42-kDa subunits. In contrast, maize

AChE exists as a mixture of disulfide-linked and non-

covalently linked 88-kDa homodimers. These results

suggest that plant AChEs exhibit various quaternary

structures, depending on the plant species, similar to

the animal AChEs (SOREQ and SEIDMAN 2001). The

full-length cDNA of siratro AChE is 1441 nucleotides,

encoding a 382-residue protein that includes a signal

peptide. Like maize AChE, the deduced amino acid se-


Characterization of the Plant Acetylcholine-Mediated System

Yoshie S. MOMONOKI and Kosuke YAMAMOTO

Graduate School of Bioindustry, Tokyo University of Agriculture,

196 Yasaka, Abashiri, Hokkaido 099-2493, Japan

62 Yoshie S. MOMONOKI and Kosuke YAMAMOTO

quence of siratro AChE exhibited no apparent similar-

ity with that of animal AChE. Based on Pfam protein

family analysis, both siratro and maize AChEs contain

a consensus sequence of the lipase GDSL family (Fig.

1), whereas the two plant AChEs do not belong to the

alpha/beta-hydrolase fold superfamily that is phyloge-

netically related to cholinesterases in animals. Genes

homologous to siratro AChE have been found only in

higher plant species.

The phylogenetic tree based on the amino acid se-

quences of AChE illustrates the clear divergence of this

enzyme family into monocots, rosid dicots, and asterid

dicots (Fig. 2). The finding that the monocot (maize)

AChE and dicot (siratro) AChE share a similar primary

structure indicates that the latest time the plant king-

dom could have acquired the common ancestor of the

plant AChE gene predates the monocot–dicot phylum

divergence event.

Further enzymatic properties of purified siratro and

maize AChEs were compared with that of electric eel

AChE. Similar to electric eel AChE, both plant AChEs

hydrolyzed acetylthiocholine (or ACh) and propionyl-

thiocholine (or propionylcholine), but not butyrylthio-

choline (or butyrylcholine), and their specificity con-

stant (kcat

/Km) was highest against ACh. There was no

significant difference between the enzymatic properties

of siratro and maize AChEs, although the plant AChEs

had low substrate turnover numbers compared with

that of electric eel AChE. The activity of the two plant

AChEs was not inhibited by excess substrate concentra-

tions, similar to butyrylcholinesterase (BChE) in ani-

mals. Thus, siratro and maize AChEs showed enzymat-

ic properties of both animal AChE (E.C. 3.1.1.7) and

animal BChE (E.C.3.1.1.8). However, both siratro and

maize AChEs exhibited low sensitivity to the AChE-

specific inhibitor neostigmine bromide, unlike other

plant AChEs. These differences in enzymatic proper-

ties among plant AChEs may reflect the phylogenetic

evolution of AChEs.

Subcellular localization of overexpressed maize

AChE in transgenic rice plants

Recombinant maize AChE was purified from

Escherichia coli Rosetta-gamiB (DE3) pLysS cells

overexpressing the maize AChE cDNA. The maize

Fig. 1. Alignment of the amino acid sequences of siratro and maize AChEs. Conserved amino

acid residues are highlighted in black. Residues not identical but similar to the conserved

ones are highlighted in gray. Blocks of sequences conserved in GDSL-family enzymes and

putative catalytic triad Ser/Asp(Glu)/His residues in the GDSL-family enzymes (reported

by BRICK et al. 1995; UPTON and BUCKLEY 1995; AKOH et al. 2004) are indicated by boxes

and arrowheads, respectively.

63THE PLANT ACETYLCHOLINE-MEDIATED SYSTEM

AChE antibody was generated using purified recombi-

nant maize AChE protein as antigen. Maize AChE was

overexpressed in transgenic rice plants, and the subcel-

lular localization of maize AChE in the rice plants was

observed by immunofluorescence.

The transgenic rice plants exhibited high AChE

activity and expression levels compared with control

plants transfected with the p2K-1+ vector only. The

AChE protein was detected in extracellular spaces in

leaf and stem tissues, suggesting that the ACh-mediated

system might function within extracellular spaces (Fig.

3; YAMAMOTO and MOMONOKI 2008).

Fig. 2. Phylogenetic tree derived from amino acid sequences of siratro AChE homologs, which were

identified by a tBLASTn analysis against TIGR gene indices. The scale bar indicates the

number of amino acid substitutions. The phylogenetic tree was constructed by the neighbor-

joining method. Numbers at branches indicate bootstrap values. The amino acid sequence

identities (%) between siratro AChE and each gene product are indicated in parentheses. The

tentative consensus (TC) numbers and GenBank accession numbers used in the analysis are

as follows: cotton, CGI TC68238; Arabidopsis, AtGI TC312464; Nicotiana, NbGI TC9050;

tomato, LeGI TC175087; wheat, TaGI TC254536; rice, OsGI TC331846; sorghum, SbGI

TC95805; maize AChE, AB093208; barley, HvGI TC146608; electric eel AChE, AF030422.

Fig. 3. Subcellular localization of maize AChE in leaf and stem tissues of transgenic rice plants.

Each section was probed with maize AChE antibody and then visualized with Alexa Flu-

or 488–conjugated secondary antibody. Control rice plants were transfected with p2K-1+

vector only. Arrowheads indicate localization of maize AChE. Scale bar, 10 µm.


Conclusion

By identifying plant AChE genes and generating

transgenic plants, this study provides the first direct

evidence of the function of ACh-mediated systems

in plants, which has remained unclear for more than

30 years. Our group has also been using chemical as-

says and histochemistry to study the responses of plant

AChEs to heat and salt stresses (MOMONOKI and MO-

MONOKI 1991; MOMONOKI 1997), and these findings

indicate (1) siratro AChE is a disulfide-linked 125-kDa

homotrimer consisting of 41- to 42-kDa subunits, (2)

Plant AChEs from maize and siratro possess enzymat-

ic properties of both animal AChE (E.C.3.1.1.7) and

animal BChE (E.C.3.1.1.8), (3) maize AChE localize

at extracellular spaces in the leaf and stem tissues of

transgenic rice plants, suggesting that the plant AChE

might regulate the opening and/or closing of channels

at the cell wall matrix.

Summary

In this study, an AChE from siratro was purified,

cloned, and characterized. The full-length cDNA of sir-

atro AChE is 1441 nucleotides and encodes a 382-resi-

due protein that includes a signal peptide. This AChE

is a disulfide-linked 125-kDa homotrimer consisting of

41- to 42-kDa subunits, in contrast to the maize AChE,

which exists as a mixture of disulfide-linked and non-

covalently linked 88-kDa homodimers. These different

quaternary structures between maize and siratro AChEs

imply that AChEs in different plant species might have

various subunit arrangements as well, similar to animal

AChEs. The enzymatic properties of siratro AChE were

compared with those of maize and electric eel AChEs.

The siratro and maize AChEs shared enzymatic proper-

ties with both animal AChE and animal BChE.

The subcellular localization of maize AChE in

transgenic rice plants was observed by immunofluores-

cence, and the maize AChE protein was detected in ex-

tracellular spaces in the leaf and stem tissues. Therefore,

the ACh-mediated system might regulate the opening

and/or closing of channels at the cell wall matrix.

Acknowledgements

This research was supported by “Ground-based

Research Program for Space Utilization” promoted by

the Japan Space Forum. The author is grateful to Dr. K.

Shimamoto (Nara Institute of Science and Technology,

Japan) for the kind gift of the p2K-1+.

References

1. AKOH, C.C., LEE, G.C., LIAW, Y.C., HUANG, T.H., and

SHAW, J.F. (2004). GDSL family of serine esterases/li-

pases. Prog. Lipid Res. 43: 534-552.

2. BRICK, D.J., BRUMLIK, M.J., BUCKLEY, J.T., CAO, J.X.,

DAVIES, P.C., MISRA, S., TRANBARGER, T.J., and UPTON,

C. (1995). A new family of lipolytic plant enzymes with

members in rice, arabidopsis, and maize. FEBS Lett.

377: 475-480.

3. HORIUCHI, Y., KIMURA, R., KATO, N., FUJII, T., SEKI, M.,

ENDO, T., KATO, T., and KAWASHIMA, K. (2003). Evolu-

tional study on acetylcholine expression. Life Sci. 72:

1745-1756.

4. KAWASHIMA, K., MISAWA, H., MORIWAKI, Y., FUJII, Y.X.,

FUJII, T., HORIUCHI, Y., YAMADA, T., IMANAKA, T., and

KAMEKURA, M. (2007). Ubiquitous expression of acetyl-

choline and its biological functions in life forms without

nervous systems. Life Sci. 80: 2206-2209.

5. MOMONOKI Y.S. (1992). Occurrence of acetylcholine-

hydrolyzing activity at the stele-cortex interface. Plant

Physiol. 99: 130-133.

6. MOMONOKI, Y.S. (1997). Asymmetric distribution of

acetylcholinesterase in gravistimulated maize seedlings.

Plant Physiol. 114: 47-53.

7. MOMONOKI, Y.S., and MOMONOKI, T. (1991). Changes

in acetylcholine levels following leaf wilting and leaf re-

covery by heat stress in plant cultivars. Jpn. J. Crop Sci.

60: 283-290.

8. ROSHCHINA, V.V. (2001). Neurotransmitters in Plant

Life. Science Publishers Inc., Enfield, pp 99-150.

9. SAGANE, Y., NAKAGAWA, T., YAMAMOTO, K., MICHIKA-

WA, S., OGURI, S., and MOMONOKI, Y.S. (2005). Molecu-

lar characterization of maize acetylcholinesterase: a nov-

el enzyme family in the plant kingdom. Plant Physiol.

138: 1359-1371.

10. SOREQ H, SEIDMAN S (2001). Acetylcholinesterase - new


roles for an old actor. Nat. Rev. Neurosci. 2: 294-302.

11. UPTON, C., and BUCKLEY, J.T. (1995). A new family of

lipolytic enzymes? Trends Biochem. Sci. 20: 178-179.

12. WIŚNIEWSKA, J., and TRETYN, A. (2003). Acetylcho-

linesterase activity in Lycopersicon esculentum and its

phytochrome mutants. Plant Physiol. Biochem. 41: 711-

717.

13. YAMAMOTO, K., and MOMONOKI, Y.S. (2008). Subcel-

lular localization of overexpressed maize AChE gene in

rice plant. Plant Signal. Behav. 3(8): 576-577.

14. YAMAMOTO, K., OGURI, S., and MOMONOKI, Y.S. (2008).

Characterization of trimeric acetylcholinesterase from a

legume plant, Macroptilium atropurpureum Urb. Planta

227: 809-822.

15. YAMAMOTO, K., OGURI, S., CHIBA, S., and MOMONOKI,

Y.S. (2009). Molecular cloning of acetylcholinesterase

gene from Salicornia europaea L. Plant Signal. Behav.

4(5): 361-366.


　本研究において，最初に siratro AChEを精製およびクローニングし，その分子特性を検討した。Siratro AChEの全 cDNA配列は，1441bpであり，シグナルペプチドを含む 382残基のタンパク質をコードしていた。本 AChEは，41－ 42-kDaの単量体がジスルフィド結合した 125-kDaのホモ ₃量体であった。対照的に maize AChEは，ジスルフィド結合および非共有結合からなる 88-kDaのホモ ₂ 量体であった。そのため，植物 AChEは動物 AChEに類似し，植物種に依存した多様な

４次構造で構成されることが推測された。さらに，siratro AChEの酵素特性を maize AChEおよび electric eel AChEと比較検討した。Siratro および maize AChEsは，動物 AChEおよび動物 BChE

双方の酵素特性を示した。次に，形質転換イネにおける maize AChEの細胞内局在性を蛍光免疫染色法により観察した。Maize AChEタンパク質は，形質転換イネの葉および茎組織の細胞外領域に検出され，植物 ACh系が細胞外領域で機能していることが示唆された。

植物アセチルコリン系の特性

桃木　芳枝・山本　紘輔

東京農業大学大学院生物産業学研究科

北海道網走市八坂 196


質疑応答司会：ご講演にあったサイラトロというのは，ちょっとご存じない方が多いかもしれませんが，熱帯のマメ科牧草でつる性のものでして，オーストラリアとか，石垣島辺りで利用されています。ただ今のご講演に対しまして，ご質問がありましたら，よろしくお願いします。ではお名前と所属と。深城：神戸大学の深城です。最後のところの重力応答のところ，非常に面白かったですけども，ノックアウトっていうか，発現を落とした１の方ですよね。屈曲反応が遅かったようでが，あのときは，例えば重力性っていうとオーキシンがやっぱり重要だと思われていますけれども，オーキシンの不等分布も影響しているのでしょうか。桃木：はい，アセチルコリンエステラーゼ遺伝子をノックダウンした抑制体の屈性は遅く，オーキシンの輸送に影響されていると考えています。オーキシンの不均等分布については，Bandurski教授とのポスドク時代，およびNASAの研究員時代に行った実験で証明しています。アイソトープ実験でもオーキシンの不均等分布は認められました。もちろん，オーキシンのみでなく，イオン類も不均等分布することが報告されています。深城：ああ，そうですか。桃木：はい。深城：はい。分かりました。ありがとうございました。桃木：それから言い忘れましたが，最後の重力応答の実験は T1種子を用いました。現在，沖縄県石垣島にあります国際農林水産業研究センターにお願いして，T2および T3世代の育成しホモライン化しています。今回，T2世代の植

物体を使った熱ストレス実験のデータを加えたかったのですが間に合いませんでした。中川先生を喜ばせることが出来ずすみません。現在，集中的に塩ストレス，熱ストレスに対する植物アセチルコリン系の応答反応について分子レベルで究明しています。良い結果が得られれば応用への発展も期待できます。今回は残念ですが，基礎研究でお許し戴きました。司会：そのほか，何かご質問ありましたら。中川：やっぱりオーキシンとアセチルコリンの関係と申しますか，メカニズム的なところですね。桃木：重力ストレス下の植物におけるホルモンの不均等分布とアセチルコリン系との関係は，次のように説明できます。屈性実験は，暗発芽させたトウモロコシ幼苗です。植物体に垂直状態から水平状態に移動する重力ストレスを与えた場合，トウモロコシ幼苗は重力屈性を示し，中胚軸の底部は上部より，オーキシン量が多く，アセチルコリンエステラーゼ活性も高くなり，双方が不均等分布を示します。伸長の程度を見ると底部は上部より大きく伸長しており。これも不均等な伸長です。一方，アセチルコリンエステラーゼ活性を阻害する臭化ネオスチグミンで植物体を処理して，重力ストレスを与えると，植物体の屈性は起こらず，組織化学的にもオーキシンの不均等分布は認められませんでした。もちろんアセチルコリンエステラーゼ活性は阻害されて検出できませんでした。これらの現象は，オーキシンの輸送にアセチルコリン系が関与していることを示しています。司会：そのほか何かご質問とか。では，桃木先生ありがとうございました。桃木：どうもありがとうございました。司会：では。（拍手）

69

Introduction

Deinococcus radiodurans is a small, red-pigment-

ed, non-spore-forming Eubacterium (Fig. 1). Members

of this species inhabit a wide range of terrestrial and

aquatic environments and are characterized by an ex-

ceptional capacity to survive the normally lethal DNA

damage induced by agents such as ionizing radiation,

UV radiation and desiccation. Deinococcus radiodu-

rans was first isolated in 1956 from canned meat that

had received 1.8 kGy of γ radiation regarded as typi-

cally lethal to bacteria (ANDERSON et al. 1956). Cur-

rently, exposure to up to 10 kGy of ionizing radiation

is used to sterilize foods. As in other organisms, the D.

radiodurans genome sustains over 100 DNA double-

strand breaks (DSBs) after exposure to 10 kGy of γ

radiation. DSBs are the most lethal form of DNA dam-

age. Although all living organisms possess DNA repair

mechanisms, only a few of the DSBs can be repaired

in most species. Deinococcus radiodurans is capable

of repairing the fragmented genome during post-irra-

diation incubation (COX and BATTISTA 2005). Genome

sequence analysis of D. radiodurans has revealed that

the genome encodes almost all the major prokaryotic

proteins involved in DNA repair (WHITE et al. 1999).

However, the molecular mechanisms underlying the ra-

diation resistance of this bacterium remain unclear.

Proteome and transcriptome analyses have re-

vealed that D. radiodurans efficiently coordinates its

recovery from exposure to ionizing radiation through

a complex array of DNA repair and metabolic path-

way switching (LIPTON et al. 2002; LIU et al. 2003).

However, the discovery of numerous additional irra-

diation-response genes has provided new targets for the

identification of genes critical to radiation resistance.

The extensive investigations conducted thus far provide

useful insights into the mechanisms underlying radia-

tion resistance, but a more detailed empirical explana-

tion of why D. radiodurans is so radiation resistant is

still needed. Further research based on alternative ge-

netic and biochemical approaches should help to give

a better understanding of the mechanisms involved in

DNA repair (NARUMI 2003).

Discovery of a Novel DNA Repair-related Protein

To elucidate the efficient DNA repair mecha-

nisms of D. radiodurans, I and my colleagues at Japan

Atomic Energy Agency have, over a 15-year period,

analyzed the mutations in the genes of DNA-repair-de-

ficient strains. The strains analyzed so far are listed in

Table 1.

Analysis of the radiosensitive strain KH311 of D.


Survival Strategy of a Radioresistant Bacterium: a Review

Issay NARUMI

Quantum Beam Science Directorate, Japan Atomic Energy Agency,

1233 Watanuki, Takasaki, Gunma 370-1292, Japan

Fig. 1 Electron microscope image of Deinococcus radio-

durans (Courtesy of H. WATANABE).

70 Issay NARUMI

radiodurans identified the absence of a novel DNA-

repair-promoting protein, PprA (pleiotropic protein

promoting DNA repair), which produced the loss of

radiation resistance. Investigation in vitro showed that

PprA protein became preferentially bound to double-

stranded DNA carrying strand breaks, inhibited E.

coli exonuclease III activity, and stimulated the DNA

end-joining reaction catalyzed by ATP-dependent and

NAD-dependent DNA ligases. These results suggest

that D. radiodurans has a non-homologous end-join-

ing (NHEJ) repair mechanism in which PprA plays a

critical role (NARUMI et al. 2004). This type of path-

way may be error-prone, because DNA ends produced

by irradiation probably undergo clustered damage, the

removal of which can create mutations. Therefore, the

NHEJ pathway must be accompanied by a mechanism

that prevents mutations to achieve accurate DSB repair

in D. radiodurans (NARUMI 2003). This mechanism

requires clarification to better understand the mecha-

nisms involved in DNA repair.

DNA ligase is one of the most frequently used

reagents in genetic engineering. Discovery of PprA

revealed the potential for a new biotech reagent from

a combination of DNA ligase and PprA. As a result

of technology-transfer by the Japan Atomic Energy

Agency to the private sector, the TA-Blunt Ligation

Kit was released in Japan as a commercial product in

November 2005. The inclusion of the PprA technology

in the ligation kit provides 10-fold increase in ligation

efficiency compared with that of conventional products

[http://www.jaea.go.jp/english/news/p06020901/index.

shtml].

Radiation Response Mechanism

The highly efficient DSB repair process in D. ra-

diodurans is radiation-inducible and is dependent on de

novo protein synthesis following irradiation (KITAYAMA

and MATSUYAMA 1968). It has been shown that both

PprA and another D. radiodurans protein, RecA, are

radiation-inducible (LIU et al. 2003). It appears that D.

radiodurans possesses a novel DNA-damage response-

mechanism. In Escherichia coli, RecA and LexA play

important roles in the DNA-damage response repair-

mechanism (the SOS system) (WALKER, 1984). In E.

coli, RecA is activated by DNA damage to mediate

proteolytic cleavage of the E. coli LexA repressor, re-

sulting in derepression of the SOS regulon. SOS-like

processes have been conserved in a wide variety of eu-

bacterial species (MILLER and KOKJOHN 1990). Neither

LexA1 nor LexA2 of D. radiodurans was found to be

involved in the DNA-damage response repair-mecha-

nism, although RecA was the sole protein required for

cleavage of the LexA1 and LexA2 proteins in D. radio-

durans (NARUMI et al. 2001; SATOH et al. 2006).

Analysis of the radiosensitive strain KH840 of D.

radiodurans identified the absence of a novel regula-

tory protein, PprI (inducer of PprA), which is involved

in the induction of PprA. Inactivation of PprI resulted

in a loss of PprA and RecA induction (HUA et al. 2003).

PprI therefore appeared to play a critical role in trig-

gering the DNA damage response and cellular survival

network following irradiation in D. radiodurans (HUA

et al. 2003).

In research in which the author was involved,

OHBA et al. (2005) identified the radiation-responsive

Table 1. The DNA repair–deficient strains of Deinococcus radiodurans that were analyzed.

Strain Gene Mutation type (genotype) Reference

KH311 pprA Base substitution (pprA446) NARUMI et al. (2004)rec30 recA Base substitution (recA670) NARUMI et al. (1999)KI696 recA Base substitution (recA424) SATOH et al. (2002)KH840 pprI IS insertion (pprI307::IS8301) HUA et al. (2003)KH586 recN 1-bp insertion FUNAYAMA et al. (1999)UVS9 uvde Base substitution (uvde335) KITAYAMA et al. (2003)262 uvrA IS insertion (uvr2230::IS2621) NARUMI et al. (1997)302 uvrA 144-bp deletion NARUMI et al. (1997)

71SURVIVAL OF RADIORESISTANT BACTERIUM

minimal promoter region of the pprA gene and demon-

strated that up-regulation of pprA expression by PprI

is triggered at the promoter level. However, we were

unable to find evidence to support direct interaction of

PprI with this promoter region. This result suggested

the existence of hitherto unknown components in the

PprI-dependent response to radiation stress in D. ra-

diodurans. In an effort to explore this possibility, the

two-dimensional protein profiles of wild-type and pprI

disruptant strains were compared (OHBA et al. 2009). In

the course of this investigation, a 10-kDa protein spot

was identified during isoelectric focusing analysis, the

isoelectric point of which differed between wild-type

and pprI disruptant strains (Fig. 2). The protein spot in

the wild-type strain indicated higher basicity than that

of the pprI disruptant strain, suggesting that the protein

may undergo post-translational modification via PprI.

We designated this protein PprM (modulator of the

PprI-dependent DNA damage response, for the reason

that follows). To determine whether PprM is respon-

sible for the radiation resistance of D. radiodurans, a

pprM disruptant strain was generated by direct inser-

tional mutagenesis using double-crossover recombina-

tion. The pprM disruptant strain exhibited markedly

higher sensitivity to γ-rays than the wild-type (Fig.

3), suggesting that PprM plays an important role in the

pprI-dependent radiation response in D. radiodurans

(OHBA et al. 2009).

To determine whether PprM is involved in the

induction of PprA and RecA, changes in the intracel-

lular levels of PprA, RecA and PprI following irradia-

tion were investigated. Constitutive production of PprA

at an elevated level was observed in the mock-irradi-

ated pprM disruptant strain, while the level of PprA

was comparable to that observed in irradiated cells of

wild and pprM disruptant strains. On the other hand,

induction of RecA was not affected by pprM disrup-

tion. These results suggest that PprM is involved in re-

pressing the production of PprA, but not that of RecA.

We proposed that only the basic form of PprM can be

involved in reversing the repression of PprA produc-

tion following irradiation in D. radiodurans (OHBA et

al. 2009).

Loss of PprA renders D. radiodurans sensitive to

radiation (HUA et al. 2003; NARUMI et al. 2004). On the

other hand, the pprM disruptant strain, which produced

high amounts of PprA in the absence of irradiation,

also exhibited high sensitivity to radiation. Two pos-

sible explanations could account for the radiosensitivity

of the pprM disruptant strain: (i) a defect in the pre-

cisely timed induction of PprA following irradiation,

or (ii) a defect in the regulation of hitherto unknown

Fig. 2 Two-dimensional PAGE analysis of Deinococcus

radiodurans wild and pprI disruptant strains. Based

on the publication of OHBA et al. (2009). Arrows

indicate a 10-kDa protein spot, the isoelectric point

of which differed between wild and pprI disruptant

strains.

Fig. 3 Survival curves of wild and gene-disruptant strains

of Deinococcus radiodurans in response to dosage

of ionizing radiation. Based on the publication of

OHBA et al. (2009). Open circles, wild-type strain;

filled squares, pprM disruptant strain; filled tri-

angles, pprA disruptant strain; filled circles, pprA–

pprM double-disruptant strain.

72 Issay NARUMI

protein(s) that are necessary for radioresistance in ad-

dition to PprA. The first explanation is supported by

previous experiments demonstrating that the lexA2 dis-

ruptant strain, in which enhancement of pprA promoter

activation was observed following irradiation, exhib-

ited much higher resistance to radiation than the wild

strain (SATOH et al. 2006). In order to confirm the latter

possibility, a pprA–pprM double-disruptant strain was

constructed and the survival rate was examined. The

pprA–pprM double-disruptant strain exhibited much

higher sensitivity to radiation than either the pprA or

the pprM single disruptant strain (Fig. 3). These studies

strongly suggest that PprM is involved in the unique

radiation response mediated by PprI and plays a crucial

role in the induction of PprA (OHBA et al. 2009). At

the same time, it was also revealed that PprM regulates

other hitherto unknown proteins important for radio-

resistance, besides PprA. It appears that there is still

much to learn about D. radiodurans.

Future Prospects

More than 40 species of the genus Deinococcus

have been discovered. The genome sequences of three of

these (D. radiodurans, D. geothermalis and D. deserti)

have been determined (WHITE et al. 1999; MAKAROVA

et al. 2007; de GROOT et al. 2009). Further comparative

genomics and molecular biological analysis involving

targeted mutagenesis and plasmid complementation

will provide new insights into our understanding of the

DNA repair mechanisms and radioresistance in Deino-

coccus species.

Acknowledgements

This work was partly supported by a Grant-in-Aid

for Scientific Research (B) 19380054 from the Japan

Society for the Promotion of Science.

References

1. ANDERSON, A. W., NORDAN, H. C., CAIN, R. F., PARRISH,

G., and DUGGAN, D. (1956). Studies on a radio-resistant

Micrococcus. I. Isolation, morphology, cultural charac-

teristics, and resistance to gamma radiation. Food Tech-

nol. 10: 575-578.

2. COX, M. M., and BATTISTA, J. R. (2005). Deinococcus

radiodurans - the consummate survivor. Nat. Rev. Mi-

crobiol. 3: 882-892.

3. de GROOT, A., DULERMO, R., ORTET, P., BLANCHARD,

L., GUÉRIN, P., FERNANDEZ, B., VACHERIE, B., DOSSAT,

C., JOLIVET, E., SIGUIER, P., CHANDLER, M., BARA-

KAT, M., DEDIEU, A., BARBE, V., HEULIN, T., SOMMER,

S., ACHOUAK, W., ARMENGAUD, J. (2009). Alliance of

proteomics and genomics to unravel the specificities of

Sahara bacterium Deinococcus deserti. PLoS Genet. 5:

e1000434.

4. FUNAYAMA, T., NARUMI, I., KIKUCHI, M., KITAYAMA,

S., WATANABE, H., and YAMAMOTO, K. (1999). Identi-

fication and disruption analysis of the recN gene in the

extremely radioresistant bacterium Deinococcus radio-

durans. Mutat. Res. 435: 151-161.

5. HUA, Y., NARUMI, I., GAO, G., TIAN, B., Satoh, K., Kita-

yama, S., and Shen, B. (2003). PprI: a general switch

responsible for extreme radioresistance of Deinococcus

radiodurans. Biochem. Biophys. Res. Commun. 306:

354-360.

6. KITAYAMA, S., and MATSUYAMA, A. (1968). Possibility

of the repair of double-strand scissions in Micrococcus

radiodurans DNA caused by γ-rays. Biochem. Biophys.

Res. Commun. 33: 418-422.

7. KITAYAMA, S., NARUMI, I., FUNAYAMA, T., and WATA-

NABE, H. (2003). Cloning of structural gene of Deino-

coccus radiodurans UV-endonuclease β. Biosci. Bio-

technol. Biochem. 67: 613-616.

8. LIPTON, M. S., PASA-TOLIĆ, L., ANDERSON, G. A., AN-

DERSON, D. J., AUBERRY, D. L., BATTISTA, J. R., DALY, M.

J., FREDRICKSON, J., HIXSON, K. K., KOSTANDARITHES,

H., MASSELON, C., MARKILLIE, L. M., MOORE, R. J.,

ROMINE, M. F., SHEN, Y., STRITMATTER, E., TOLIĆ, N.,

UDSETH, H. R., VENKATESWARAN, A., WONG, K. K.,

ZHAO, R., and SMITH, R. D. (2002). Global analysis of

the Deinococcus radiodurans proteome by using accu-

rate mass tags. Proc. Natl. Acad. Sci. USA 99: 11049-

11054.

9. LIU, Y., ZHOU, J., OMELCHENKO, M. V., BELIAEV, A. S.,

VENKATESWARAN, A., STAIR, J., WU, L., THOMPSON, D.

K., XU, D., ROGOZIN, I. B., GAIDAMAKOVA, E. K., ZHAI,

M., MAKAROVA, K. S., KOONIN, E. V., and DALY, M. J.

(2003). Transcriptome dynamics of Deinococcus radio-

durans recovering from ionizing radiation. Proc. Natl.

Acad. Sci. USA 100: 4191-4196.


10. MAKAROVA, K. S., OMELCHENKO, M. V., GAIDAMAKOVA,

E. K., MATROSOVA, V. Y., VASILENKO, A., ZHAI, M., LAPI-

DUS, A., COPELAND, A., KIM, E., LAND, M., MAVROMMA-

TIS, K., PITLUCK, S., RICHARDSON, P. M., DETTER, C.,

BRETTIN, T., SAUNDERS, E., LAI, B., RAVEL, B., KEM-

NER, K. M., WOLF, Y. I., SOROKIN, A., GERASIMOVA, A.

V., GELFAND, M. S., FREDRICKSON, J. K., KOONIN, E. V.,

and DALY, M. J. (2007). Deinococcus geothermalis: the

pool of extreme radiation resistance genes shrinks. PLoS

ONE 2(9): e955. doi:10.1371/journal.pone.0000955.

11. MILLER, R. V., and KOKJOHN, T. A. (1990). General mi-

crobiology of recA. Environmental and evolutionary sig-

nificance. Annu. Rev. Microbiol. 44: 365-394.

12. NARUMI, I (2003). Unlocking radiation resistance mech-

anisms: still a long way to go. Trends Microbiol. 11:

422-425.

13. NARUMI, I., CHERDCHU, K., KITAYAMA, S., and WATA-

NABE, H. (1997). The Deinococcus radiodurans uvrA

gene: identification of mutation sites in two mitomy-

cin-sensitive strains and the first discovery of insertion

sequence element from deinobacteria. Gene 198: 115-

126.

14. NARUMI, I., SATOH, K., CUI, S., FUNAYAMA, T., KITAYA-

MA, S., and WATANABE, H (2004). PprA: a novel protein

that stimulates DNA ligation. Mol. Microbiol. 54: 278-

285.

15. NARUMI, I., SATOH, K., KIKUCHI, M., FUNAYAMA, T.,

KITAYAMA, S., YANAGISAWA, T., WATANABE, H., and

YAMAMOTO, K. (1999). Molecular analysis of the Deino-

coccus radiodurans recA locus and identification of a

mutation site in a DNA repair-deficient mutant, rec30.

Mutat. Res. 435: 233-243.

16. NARUMI, I., SATOH, K., KIKUCHI, M., FUNAYAMA, T.,

YANAGISAWA, T., KOBAYASHI, Y., WATANABE, H., and

YAMAMOTO, K. (2001). The LexA protein from Deino-

coccus radiodurans is not involved in RecA induction

following γ irradiation. J. Bacteriol. 183: 6951-6956.

17. OHBA, H., SATOH, K., SGHAIER, H., YANAGISAWA, T., and

NARUMI, I (2009). Identification of PprM: a modulator

of the PprI-dependent DNA damage response in Deino-

coccus radiodurans. Extremophiles 13: 471-479.

18. OHBA, H., SATOH, K., YANAGISAWA, T., and NARUMI, I.

(2005). The radiation responsive promoter of the Deino-

coccus radiodurans pprA gene. Gene 363: 133-141.

19. SATOH, K., NARUMI, I., KIKUCHI, M., KITAYAMA, S.,

YANAGISAWA, T., YAMAMOTO, K., and WATANABE, H.

(2002). Characterization of RecA424 and RecA670 pro-

teins from Deinococcus radiodurans. J. Biochem. (To-

kyo) 131: 121-129.

20. SATOH, K., OHBA, H., SGHAIER, H., and NARUMI, I.

(2006). Down-regulation of radioresistance by LexA2

in Deinococcus radiodurans. Microbiology 152: 3217-

3226.

21. WALKER, G. C. (1984). Mutagenesis and inducible re-

sponses to deoxyribonucleic acid damage in Escherichia

coli. Microbiol. Rev. 48: 60-93.

22. WHITE, O., EISEN, J. A., HEIDELBERG, J. F., HICKEY, E.

K., PETERSON, J. D., DODSON, R. J., HAFT, D. H., GWINN,

M. L., NELSON, W. C., RICHARDSON, D. L., MOFFAT, K.

S., QIN, H., JIANG, L., PAMPHILE, W., CROSBY, M., SHEN,

M., VAMATHEVAN, J. J., LAM, P., McDONALD, L., UTTER-

BACK, T., ZALEWSKI, C., MAKAROVA, K. S., ARAVIND, L.,

DALY, M. J., MINTON, K. W., FLEISCHMANN, R. D., KET-

CHUM, K. A., NELSON, K. E., SALZBERG, S., SMITH, H.

O., VENTER, J. C., and FRASER, C. M. (1999). Genome

sequence of the radioresistant bacterium Deinococcus

74 Issay NARUMI

radiodurans R1. Science 286: 1571-1577.

　生物の中でも極めて放射線に強い微生物群を総称して，放射線抵抗性細菌と呼んでいる。1956年，ガンマ線滅菌したはずの缶詰の中で増殖している細菌が見つかり，これが放射線抵抗性細菌の最初の発見となった。Deinococcusの放射線耐性機構の研究は，そのほとんどが，最初に分離された D.

radioduransを用いて行われている。電離放射線による生物効果の中で最も重篤な損傷は DNA二重鎖切断であり，放射線を照射すると，D. radio-

duransは，大腸菌の様な一般的な細菌と同程度に，ゲノム DNA内に二重鎖切断を受ける。一般的な細菌では，細胞内に生じた数個の DNA二重鎖切断による損傷が致死的効果を与えるのに対して，D. radioduransは，細胞内に生じた 100箇所以上の二重鎖切断損傷を短時間で修復することができる。すなわち，D. radioduransの放射線耐性は，この菌のもつ優れた DNA修復能に大きく依存している。我々の研究グループがとった研究戦略は，D. radioduransから分離された放射線感受性変異株の原因遺伝子を同定することであった。その結果，D. radioduransの放射線耐性に重要な新

規遺伝子を同定することに成功した。同定した遺伝子は，他の生物で解析済みのどの遺伝子とも全く似ておらず，機能未知遺伝子に分類されていたもののひとつであった。この遺伝子から作られるタンパク質 PprAの性質を解析した結果，放射線照射後の細胞内で生合成が活発になり，放射線によって DNA鎖が切れた部分を認識して結合することにより，DNA鎖切断の修復を高効率で促進する作用をもつことが分かった。PprAは放射線誘導性タンパク質であるが，D. radioduransの放射線応答機構にもユニークなタンパク質群が関与していた。我々の研究から，DNA修復タンパク質の放射線誘導制御に係わる新規因子 PprI及びPprMが同定されたが，これらの因子によって制御を受けるまだ未知の重要な DNA修復関連タンパク質の存在が示唆される。現在，Deinococcus

属細菌の 3菌種についてゲノム配列が解読されているが，放射線抵抗性細菌の放射線耐性の共通原理を解明するためには，さらなる比較ゲノム解析と分子生物学的解析が必要である。

放射線抵抗性細菌の生存戦略

鳴　海　一　成

独立行政法人日本原子力研究開発機構　量子ビーム応用研究部門

バイオ応用技術研究ユニット

〒 370－1292　群馬県高崎市綿貫町 1233


質疑応答司会：では質疑応答に移りたいと思います。ご質問ございますか。どうぞ。風間：理化学研究所の風間と申します。最初のスライドの方で ₂ kGy照射しても４時間後にはゲノムが元通りに戻ってしまうがすごく印象的でした。exonucleaseを阻害して，DNA ligaseの活性を促進するという PprAタンパク質の特性から考えると，高等生物の end joiningみたいなのが，すごく促進されるのかなと。そうすると元通りになる機構っていうのは，end joining

の活性が強いからだと思いますが，その辺はどういう機構があると考えられているのでしょうか。鳴海：DNA ligaseの活性を促進するというのは，真核生物の Kuタンパク質と DNA-PKcsの系，それから Mre11/Rad50/Xrs ₂ タンパク質複合体の系の働きにそっくりなのです。ですから，non-homologous end joining経路のバクテリア版を，この PprAが ₁ つのタンパク質でやっているのかもしれないと思います。真核生物の Ku

タンパク質のホモログが，枯草菌とかマイコバクテリアには存在しますが，これらはアミノ酸配列が保存されている本当のKuホモログです。一方，PprAタンパク質は，Kuホモログともアミノ酸配列が全然似てないので，全く新しいものだと思いますが，end joiningの DNA修復機構には係わっているだろうと考えています。homologous recombinationとの連携については，RecAタンパク質など homologous recombination

に関係するようなタンパク質と PprAタンパク質を掛け合わせてどうなるかという実験を行いましたが，ものすごく recombination活性が上がったという結果は得られていませんので，PprAタンパク質と homologous recombinationとの関係については，まだちょっと分かりません。風間：ありがとうございます。司会：ほかにございませんか。じゃあ中川さん。中川：生物資源研究所の中川です。今の質問と関係しますが，ゲノムが何ヶ所も切れて，それが元通りに戻るというところが，非常に不思議に思います。先ほどちょっと話があったネムリユ

スリカも乾燥していくと，染色体がばらばらになりますが，それを吸水させると，ちゃんと元どおりに修復されると聞いています。この場合，ばらばらになった染色体がいったいどのように修復されたら，逆位や転座などの染色体異常が起こらないだろうかと不思議に思っています。染色体異常が起こらない様に何か制御しているメカニズムがあるのでしょうか。鳴海：バクテリアのゲノムっていうのは，核膜がありませんが，核様体という DNAと DNA結合タンパク質の複合体構造があって，細胞内にあまり散在しないような状態にあります。そうすると DNAの double strand breakができても，切れた DNA断片が散在せずに，DNA末端の切れ口同士が近くに存在していることになるので，このことが染色体異常の起こらないことと関係するという説もあります。ただ，放射線抵抗性細菌では，DNA結合能を持つ核タンパク質をコードするある種の遺伝子を破壊すると，細胞の中に DNAが散在しますが，その様な遺伝子破壊株の放射線耐性は野生株と同様だったという実験結果もありますので，染色体構造の特異性と染色体異常抑制機構の関係についてはまだ分からないことが多いと思います。谷坂：京大の谷坂です。DNA型のトランスポゾンの切り出しは，恐らく放射線と同じような切断を受けて修復すると思いますが，その修復にも同じような機構が働くと考えていいのでしょうか。鳴海：放射線による DNAの切断点は，制限酵素などの酵素反応で切ったようにきれいな切り口ばかりではありません。5’末端にリン酸がついていたりいなかったり，様々な，いわゆる汚い切れ方があると思います。このような切断点を修復する際に，単純に DNA ligase

で繋げてしまうと，mutationが起こってしまいます。この様なことから，真核生物の non-

homologous end joiningは誤りがちな修復機構と言われています。一方，放射線抵抗性細菌には，誤りがちではない end joining機構があるのかも知れません。また，それだけでは不十分で，recombinationが最終的には重要になってくるのではないかと思っています。

76 Issay NARUMI

谷坂：ありがとうございました。司会：ほかにございませんか。では，時間もきておるようですし，立体構造までお決めになっていると，とても面白い，興味深いなと思って聞

いておりました。ますますの研究のご発展をお祈りしております。鳴海先生ありがとうございました。

（拍手）

77

司会：では総合討論をこれで始めさせていただきます。総合討論座長に北海道大学の佐野先生にお願いしております。よろしくお願いします。佐野：北海道大学の佐野です。総合討論ということで，皆さん，ご協力をお願いしたいと思います。　今年度のシンポジウムは非常に多岐にわたって，全体のタイトルが，環境耐性機構の解明と分子育種ということで，育種に関係するような重要な問題，ほとんど網羅的に取り上げられているのです。それで，それを総合的に討論するというと，どこにターゲットをしぼるかということは，非常に皆さんも迷われるところではないかと思います。多分，その点は今後，一番難しい点は，わたしは新しい分子情報とかは今まで複雑なメカニズムの解析にものすごく有効な手段を提供してくれているという点においては，多分，一緒です。それを今後，どのように実際に使っていくかというのは，このシンポジウムは基礎的な研究対象になっていますけれども，応用との研究との接点をねらうところでは，やっぱり一番問題になるのは，ここまで来ていて，どうしてもっと分子育種的なことを推進できないのか，ネックはどこなのかというところが，恐らくかなりの方が関心を持っている総合的討論のポイントだろうと思うのですね。時間があればそういうことはやっていただいていいのですが，多分そこまでの時間はないだろうと今回は思って，個別の質疑応答というところを発展させて，関連点を探っていただくというような形で進めたいと思っています。　それで大きく分けますと，必須元素の昨日からのいろいろな話題の中で必須元素の利用戦略ということで，鉄だとかホウ素に関して非常に素晴らしいご報告があったと思います。それと，もう ₁つ，吸収系の根系だとか，あるいは耐寒性という，かなり形態なんかに結びついた問題点の分子メカニズム。また，もう ₁つのグループとしては，高温不稔，穂発芽というような実際的な育種対象に

なるような形質の変化を分子レベルで。きょうのご講演では，アセチルコリンという今まであまり植物では見られなかったもので，かなり広範に非常に重要な役割をしているのではないかという問題点の提起，今後のものに，方向に期待されるという。最後はちょっと話が違うかもしれませんが，微生物での修復機構というのは，もともと，ちょっとお話があったように，昔から植物の修復機構に関しても結構仕事があって，育種の放射線，突然変異利用というのは，修復から逃れたものをいかに利用するかというような観点から，昔は随分，植物の，海外でも研究されて，勉強されたと思うのです。そういうことで，今なお，新しい大きな発見があるというようなことを今後の研究につなげるという意味で，いろいろ関連はあろうかと思います。　ちょっと前置きが長くなりましたが，そういう大きな観点から見ていただいて，まず必須元素の利用，植物の利用戦略ということで，非常に新しい方向ならびに具体的な事例が出てきたことに関して，昨日の質問ではできなかったような質疑・応答がございましたら，その点からよろしくお願いしたいと思います。西尾：東北大学の西尾でございます。　昨日，西澤先生と藤原先生にお伺いをしたいのですが，鉄の吸収およびホウ素の輸送，吸収というようなことに関して，素晴らしいご講演をいただいたのですが，今，農水プロジェクトでカドミウムの高吸収，低吸収というのを，随分たくさんのグループがやっておりまして，わたしどももそれにかかわっておるのですが。カドミウムのトランスポートに関して，こういう両先生のご研究の発展で，最近の知見とか，こういうトランスポーターが低吸収，高吸収にかかわっているとかというような情報をお持ちでしたら，教えていただければ幸いです。西澤：まず，鉄とカドミウムの関連なのですが。

総合討論

座長　佐野芳雄（北海道大学）

78

カドミウムは植物の生育にとって必須元素ではありません。けれどもカドミウムを水稲の根に与えて，PETISの手法でリアルタイムに観察しますと，₁₂時間ぐらいでもう穂に行ってしまうことが，原研の藤巻さん達の研究で明らかになっています。ということで，稲がカドミウムを根から吸収して地上部に移行させることは確かですが，カドミウムそのものは必須元素ではないので，多分，ほかの必須元素のトランスポーターのうち基質特異性の低いようなものによって，吸収されて地上部に行っていると思います。鉄とカドミウムというのは非常に関係が深くて，ちょっと話が長くなりますが，水稲でカドミウムの吸収を抑制するために，農水省の指針として出穂前後の ₃週間を湛水にしておくことが薦められています。というのは，湛水により土壌が還元状態になっているとカドミウムが不溶体となって溶け出さない。それが落水することによって酸化状態となると，カドミウムが可溶化して吸収されやすくなります。出穂期に吸収したカドミウムが穂に行ってしまうことを避けるために，出穂前後の ₃週間，湛水条件に保つということが，技術として確立されています。このカドミウムと，まったく反対の関係にあるのが必須元素の鉄で，鉄は湛水で土壌が還元状態になっていると， ₂価鉄になって溶けやすくなっているため，稲は鉄欠乏にならないのですが，落水して酸化状態になって，鉄が ₂価から ₃価になってしまうと溶けにくくなる。そういう状態では，稲は鉄欠乏を感知して，昨日お話ししましたように，₂価鉄イオンのトランスポーター IRT ₁ とか，それ以外の鉄吸収のためのトランスポーターを活性化します。吸収トランスポーターの活性化もそうですし，それから移行も活性化します。ですから落水によって稲が鉄欠乏となり，それによって発現が上昇した鉄のトランスポーターがカドミウムを吸収するのではないかという仮説を立てました。鉄欠乏によって強く発現が誘導される ₂価鉄イオントランスポーター，IRT ₁ と ₂ を単離していますが，これらがカドミウムも吸収するかどうかを酵母を使った実験で確認しました。そうしましたら，IRT ₁ も ₂ もカドミウムを吸収することがわかりました。「鉄・ムギネ酸類」錯体のトランスポーター，YSLは同様に鉄欠乏によって強

く誘導されますが，カドミウムの吸収には関与していないと思います。カドミウムとムギネ酸類は錯体を作ることは可能ですが，非常に弱いのでカドミウムの吸収にはムギネ酸類は関与していないという報告があります。 ₂価鉄イオンのトランスポーターを使う，あるいは PETISの手法でカドミウムの移行をみると，亜鉛の吸収移行とよく似ていることから，亜鉛のトランスポーターも使っていると思います。ですから，少なくとも鉄のトランスポーターの ₁部は使い，それからそれ以外のトランスポーターも使っているだろうと考えております。藤原：西尾先生のご質問を ₂通りに解釈いたします。まず，西澤先生のお答えになっていない意味合いで，ホウ素の輸送とカドミウムの輸送が関係あるのでしょうかというのは，多分ないと思います。別のとらえ方で，このような研究のアプローチでカドミウムの輸送がどの程度理解できるのでしょうかというようなお問いかけだとすれば，わたしどもの理解としては，いろんな方が，稲に限定すれば，稲で吸収量の高いもの，低いもの，もしくは耐性，わたしどもは耐性の違うような変化を対象にするというような努力をしておりまして，そういったものから原因になる遺伝子というのは，恐らく見つかってくると思います。それが，実際にどの程度有用なものかというのはまた，次の，なんていうのでしょう，いろいろな試験というものが当然，必要になってくると思うのですが，こういうアプローチで，より使える系統といいましょうか，そういったものが生まれてくるんじゃないかというふうに思っています。佐野：ありがとうございます。もう一方，谷坂先生。谷坂：京都大学の谷坂です。　西澤先生にお伺いしたいのですが，われわれ，育種をやっている者にとって，今，世界的に非常に重要なテーマというのは，乾燥耐性と，やはり酸性，アルカリ性土壌に対する耐性の品種をつくること。非常に大きな課題になっているのです。そういう点で，きのうのご講演を聴きまして，大変うれしく思ったわけなのですが，何か目が見えてきたなという感じがするのですね。そのキレート戦略をつくったアルカリ耐性の稲ですが，具体

79

的に pHがどこぐらいまでいけるのか，あるいは世界的にみますと，今の水田の中でアルカリ土壌といわれるのは大体どのくらいなのか。それと比べて，先生のおつくりになった稲がどういうふうに栽培できるか，非常に興味があるところですので教えていただきたいと思います。西澤：はい，ありがとうございます。私達が，隔離温室，それから圃場で生育検定に使った石灰質土壌の pHは約₉. ₀です。世界的にみれば，pH ₉～₁₀ぐらいの石灰質土壌というのはたくさんありますし，それからややアルカリ性が低い pH ₇ ～₈ ぐらいの土壌は，ずっと広い範囲で広がっています。私達が作った稲は pH ₉ ぐらいであれば，まったく問題なく順調に生育することができるということが言えると思います。それでよろしいでしょうか。谷坂：すごく期待できるということで，うれしく思います。ありがとうございました。西澤：ありがとうございました。佐野：ほかにございますか。それでは，またあとで全体としてのご質問をいただければいいと思います。　次の話題として出ましたのは，側根発生と耐旱の根についてです。結構，実質的なケースとして重要な，栽培上に重要なポイントも含めたお話があったと思うのですが，深城先生と犬飼先生のご講演に関して何かご質問なりありましたら，お願いしたいと思います。ございませんか。　わたしからちょっと質問させていただきたいのですが，犬飼先生のほうでもお話ししていたように，きれいなデータを出そうと思うと，非常に精密な実験系が必要だと思うんですね。スプリンクラーをつけて，勾配を。あれでもなかなか，実際に圃場を使われているから大変だと思うのですが，そういうものと実際の天水田という自然条件における，いろんな環境のふらつきなんていうものは，とんでもなく違うだろうと思うのですが，その辺のことに関しては，将来的にはどういうふうにお考えなのか。犬飼：そうですね。たしかに，あれもビニールハウスの中で，かなり違った，天然の気象とは違ったような形で育てていますので，あれがどこまで反映されているのかというのは，ちょっと疑問に

は残るところなのですが，今のところは，もう少しあれを現場に近づけるようなスクリーニングはちょっとなかなかできていないということが現実です。ただ，いろんな品種を使っていまして，実際に天水田で農家さんが，東北のタイとかに行って聞いてきたのですが，これとこれは，干ばつ年であっても，あるいは雨がいっぱい降る年であっても，そんなに収量が劇的に変わらないというふうに，好んで使っているような品種が，スクリーニングの系で強いということがありますし，おかぼの品種を育てると，あれでは強くないのです。そういうことを含めると，ある程度は天水田の栽培環境みたいなものをちゃんと反映できるような結果ができているのかなと，今のところは考えています。佐野：ほかにご質問。－－　犬飼先生のほうの話では冠根が増えると，天水田とかの乾燥耐性が増えるというお話でしたが，深城先生のほうの話として，側根というものと，乾燥耐性とかそういう，やはりストレス耐性に関する一般的な話として，なんかそういう関係がもしわかっていましたら，教えていただければと思います。深城：はい，ありがとうございます。シロイヌナズナの研究で，乾燥耐性のときに根系がどうなるかということを詳しく調べた研究というのはあまりないですね。ただ，いろいろな，例えばリン欠応答などのときに，根系が大きく変化するということがよく，ほかの植物でも知られていますが，シロイヌナズナでも，例えば培地のリン酸濃度を下げたときに，主根の伸長であるとか，側根の数がどうなるかという研究はされております。そうすると，リン欠にしますと，主根の伸長が阻害されて，側根が増えるということが何年か前に報告されていました。　最近，面白い報告があって，そのときにオーキシンの感受性を変えているんじゃないかという話が出てきまして，実際調べてみますと，リン欠にしますと，オーキシンの受容体で，きのうの発表でお話しした TIR ₁という受容体，この発現が上昇するのです。上昇することで，オーキシンの感受性を高めて側根を増やしているという話があります。したがって，これはリン欠応答の話ですが，

80

それ以外の，例えば乾燥とか，水分，要するに水ストレスですね，こういったときにも，もしかしたら内生のオーキシンの感受性であるとか，オーキシンの合成とか，そういったものを変化させて，根系パターン，側根の本数とか伸長を調節することで適応すると，そういったことが恐らくあるんじゃないかなというふうに，わたし自身は考えています。－－　ありがとうございます。佐野：ほかにございますでしょうか。オーキシンの話で（桃木）先生，何かコメントがございましたら。　じゃあ，次の，きのうの最後にお話をされました（松井）先生です。稲の高温不稔。それと，穂発芽のほうも一緒に，かなり傾向は違うのですが，お二方のご講演に関してご質問ございましたら。中川：生物研，中川です。葯の裂開の有無，花粉の散り方が高温不稔の原因という話であったと思いますが高温にあたった花粉の伸長側類というような情報があるのでしょうか？あるいは花粉の稔性や伸長には差が無く，結局花粉の散り方のみが問題ということなのでしょうか？松井：屋外で，普通に，普通にというか，高温不稔が出始めるような温度では，花粉は大抵，正常に発芽をしているんですね。正常といっても₅₀％ぐらいの発芽率が普通かなと，わたしは思うのですが。発芽のほうも観察はしているのですが，発芽が少ないから不稔になるというケースは少ないですね。まれですね。オーストラリアみたいに開花後，非常に温度が急激にさらに上がっていくというような特殊な環境，そういうところでは開花前日の高温によって花粉がもう死んじゃっているというのでしょうか，発芽しない。それが直接の不稔の原因になる場合があるみたいですが。われわれが住んでいるような湿潤なところでは，受粉がうまくいかないということが問題です。中川：花粉が粘っているとか，もちろん開くところが大きい，小さいというのが，徹底的な要因になっていると。日本の，例えば高温不稔の場合ということですか。松井：そうですね。割れ方が大きいか，小さいかというのは，これはもう，かなり遺伝的に決まっているようで，少々環境が変わっても，そんなに

変化しないのですが。重要なのは，花粉がやっぱり粘っこくなるというのはちょっと変な表現ですが，引っ付いて固まっちゃっているということです。どうして粘るのかというのは，もうひとつ，よくわからないのですが。そこのところが不稔の直接の原因かと考えています。中川：ありがとうございました。佐野：ちょっと，今のに関連して，わたしからも質問があるのですが。高温不稔と低温不稔では，葯の問題がいつも言われるのですが，花粉量が，低温の場合は葯の大きさをよく言われるのですが，その場合は裂開性のことをあまり今まで話題には出てきていないですね。高温不稔と低温不稔での，そのあたりの差というのは何かお考えはありますでしょうか。松井：あまりないですが，高温の場合もやはりやくが大きいと強いのではないかというのは，わたしも最初考えて，いろいろと試してみたのですが，平均値，全体としてどれだけ花粉が落ちているかというより，そのばらつき，そちらのほうが重要な感じです。一部の花にたくさん花粉が落ちていてもしょうがないので。佐野：相関がないということ。松井：あまり葯の大きさと高温耐性との相関は小さいです。佐野：低温の場合は，はっきり相関が出ますから。松井：そうですね。佐野：ですね。それがはっきりしているのですね。どうも，ありがとうございました。ほかにございますでしょうか。きょう，お話をいただいた杉本先生は非常に幅広い内容なのですが，何か。藤野：ホクレンの藤野です。　きょうの Sdr ４のほうで，栽培稲のコレクションでアプロタイプを調べると， ₃ 種類しかないと。イメージとしては，これはコレクションなので，それなりにバリエーションがあって，そういうのを探索すると育種的にいいアリルを使ってこれるんじゃないかなというイメージがあるのですが，この遺伝子が機能的に大事なので ₃種類しかないのか，それともコアコレクション的にみると，そんなにいっぱい自然変異としてアリルみたいなものがないのかというのは，どういう感じなんでしょうか。

81

杉本：質問ありがとうございます。　 ₃種類しかなかったのは本人も驚いていて，コアコレクションをよむことによってバリエーションがあって，どれがＦＮＰなのかわかるようなことを期待していたのですが，調べると ₃種類しかなくて，そのうち ₂種類は野生にでも存在したので，非常にワイルドなまま使っている遺伝子なんだなという印象を受けています。　それで，ちょっとちゃんと答えになっているかはわからないのですが，少し Sdr ４の機能が頃合いに落ちたものが，日本晴型の Sdr ４で，それで十分だったのか，地域の栽培の形とか，地域の湿度とか，いろいろ条件があると思いますので，ある程度の幅の範囲で休眠性が変わっていて，その範囲では ₂つで，あるいは ₃つで十分，対応できたのかなと。今，想像しているだけなのですが，なかなか直接お答えできるような考えはまだまとまっていません，残念ながら。西尾：Sdr ４の変異体が，胚（はい）が大きくなっているというのは興味深かったのですが，今，巨大胚（はい）の変異体が随分，新形質の米として注目されていて，たくさん，品種，系統が出てきたりしているのですが，巨大胚（はい）変異の中で，この Sdr ４の突然変異というようなものは調べておられるのでしょうか。杉本：大きな胚（はい）の品種は，興味があったのですが，まだ Sdr ４がどうなっているかを見たことがありません。ただ，sdr ４の切片，あまりいい切片ではなかったのですが，かなりバランスよく胚（はい）が大きくなっていて，例えば長戸先生のところの GiantEmbryoのようにスキューテラムがすごく大きくなっているとか，巨大胚（はい）で有名な品種はやはりスキューテラムのところがすごく大きくなって，種をカバーしてしまうような形になっていますが，その点，Sdr ４のミュータントは，なんとなく止まらなくて，発達がズルズル，成長がズルズル起こってしまっているというような印象なので，若干，巨大胚（はい）として報告されているものとは違うものではないかなという印象を受けています。佐野：それでは，きょうのご講演をいただきました，まず桃木先生のほうのアセチルコリンのお話で，何か追加のご質問がございましたら。

鳴海：最後の仮説のところで，アセチルコリンレセプターがあって，その中をいろんな物質が通っていくような感じがあったのですが，そういうのは動物では，やはりそういうようなこと解っているのでしょうか。植物ではアセチルコリンレセプターも，もうわかっているのでしょうか。桃木：動物のアセチルコリンは神経伝達物質としてよく知られ，アセチルコリンレセプターも見つかっています。また，よく研究されています。植物ではレセプターそのものはまだ同定されていませんが，レセプター様タンパク質は見つかっております。その解明にはまだ時間がかかると思います。しかし，最近は機器の性能も進歩していますので短期で済むかもしれません。動物の場合はニコチン性とムスカリン性の ₂種類のレセプターがあります。刺激による敏速な反応を示す電気ウナギや電気ナマズの発電器官や交感神経終末などはニコチン性レセプター，一方副交感神経終末，汗腺の一部，脊髄の運動や感覚の神経伝達にはムスカリン性レセプターが関係します。植物にもレセプター様タンパク質が動物と同じように ₂種類あると報告されていますので，動物と植物はある程度作用が似ていると推定されます。しかし，アセチルコリンエステラーゼのインヒビターに対する感受性のオーダーが ₂桁位違いますので，反応速度に大きな差異があると考えています。鳴海：それと，植物アセチルコリンエステラーゼは動物アセチルコリンエステラーゼより活性が低い。植物にレセプターがあると考えられるけど，まだわかっていない。本当に活性が低いのですか。桃木：植物アセチルコリンエステラーゼの ₁分子あたりの活性は確かに動物アセチルコリンエステラーゼよりも低いです。レセプターに関しては同定されていないものの，細胞膜にアセチルコリンが結合する等の植物アセチルコリンレセプターの存在を示唆する報告があります。それから，アセチルコリン系が作用するところでは，まずアセチルコリンがレセプターに結合し，その結合によってチャネルが開き物質が通過する作用が起きると仮定されます。その後はレセプターに結合したアセチルコリンはアセチルコリンエステラーゼに分解されます。すなわち作用の起こる部位では，必ずアセチルコリンエステラーゼの活性に変動が見

82

られると推測できます。それ故，我々はアセチルコリンエステラーゼに注目，タンパク質を精製しその一次構造を決定し，さらにタンパク質をコードする遺伝子を単離しました。このトウモロコシアセチルコリン遺伝子（maize AChE）をイネに導入して，過剰発現体を作出し，イネの葉や茎における maize AChEの細胞内局在性を蛍光免疫染色法にて観察しても，さらに GFP遺伝子を融合させた maize AChE遺伝子をイネに導入して観察しても maize AChEは細胞外領域に局在することが認められました。これらの結果らもアセチルコリンレセプターの存在が示唆されます。　植物アセチルコリン系の研究は₃₈年経て， ₃分子から構成されるアセチルコリン系の ₁つであるアセチルコリンエステラーゼについて，ようやく分子レベルでの研究が始まったばかりです。レセプターについてはこれからです，佐野：ありがとうございます。ほかにございますか。　それでは，きょう最後のご講演になりました，鳴海先生にご質問のある方，お願いしたいと思いますが。久保山：茨城大の久保山です。やっぱりちょっと気になるのは，なぜこのように放射線に強いというか，修復機構が極端に発達したかということですね。そういう必然性というか，必要があったのかどうかという話。もし考えがあったら，教えてください。鳴海： ₂つ仮説があります。　 ₁つは，アメリカのグループが提唱しているのですが，乾燥適応仮説。進化の過程で乾燥に強くなるメカニズムを得た副産物として，放射線にも強くなったという仮説です。これは，乾燥でもDNAの double strand breakが起こるので，それに効率的に対処するために適応して，その結果，放射線にも強くなったという仮説です。　もう ₁ つは，放射線適応仮説です。微生物は₂₀億年位前にすでに基本的な DNA修復機構を獲得しています。一方，₂₀億年前の地球上には天然原子炉が存在していたということが分かっています。ウラン鉱床で自然的に臨界状態が持続して，地下の数キロの海中で自然の核分裂が起こっていた時期というのがあるのです。そこには当然，微

生物がいて，₁₀₀万年位の間，天然原子炉の臨界状態が続きます。大体，₆₀Gy/h程の線量率と推定されていますが，天然原子炉からの放射線に適応するために微生物の進化が起こったということも考えられます。実際に，深海の熱水鉱床近辺というのは，昔，天然原子炉が稼働していたところでありますし，深海にいる超好熱性の微生物も放射線に強いという事実があります。ウランの半減期を考えるとすぐわかりますが，天然原子炉は₁₇億年前には稼働しなくなります。その後，放射線耐性に進化した遺伝子がどうやって今まで残ってきたかというのは，乾燥でもいいのではないかなというような仮説も成り立つ訳です。久保山：ありがとうございます。佐野：ほかにございませんか。鵜飼：開発された DNA修復試薬は，植物細胞とか動物細胞には有効なのでしょうか。鳴海：pprA遺伝子を動物や植物の中に入れて働くかどうかというご質問と考えていいですか。動物では培養細胞に核移行シグナルを付けた pprA

遺伝子を導入した例はあるのですが，なかなかやはりうまく機能しません。動物細胞にはそれ自身のDNA修復機構がありますから，それとバッティングする様です。また，pprA遺伝子が DNAに傷ができたときだけ働くような工夫をすることも必要です。最初から pprA遺伝子が細胞内で高発現していると，DNAの複製に悪さをしたりしますので，そういう制御が全部できた上で，他の生物に導入すると pprA遺伝子がうまく働く細胞系が作れるかもしれません。植物については，まだやったことがありません。佐野：よろしいでしょうか。もう少し時間があるのですが。中川：生物研，中川です。　放射線感受性の話をすると，鵜飼先生も興味があると思うのですが。放射線に強い生物でも突然変異率が上がっていくのか，あるいは例えばLET が₁₀₀ keV/µmのところが一番変異率が高くなってしまうのか。放射線抵抗性は突然変異率にどう影響するか。鳴海：₁₉₇₀年代，₈₀年代に，放射線抵抗性細菌デイノコッカス・ラジオデュランスの突然変異率を調べた研究があります。その論文では，生存率が

83

₁₀％や ₁％に低下する様な線量を照射しても，突然変異率は一定であるというデータが出ていたのですが，この結果に疑問を持って再試をしたフランスの研究者がいます。抗生物質耐性の変異コロニーの出現頻度を指標にして突然変異率を算出するのですが，再試の結果，線量を上げていくにしたがって，突然変異率が上がるという結果でした。₁₀倍程度までは上がります。しかし，放射線抵抗性細菌は，大腸菌の様に，SOS修復応答で誘導される誤りがちな DNAポリメラーゼを持っていないので，突然変異を積極的に誘導するようなメカニズムは持っていません。その点では，放射線抵抗性細菌では突然変異が起こりにくいと言えますが，一般的に，放射線に強い生物でも，弱い生物でも，誤りがちなポリメラーゼを持っているかどうか，それから mismatch repairなどの DNA修復機構が完全であるかどうかによって，突然変異率が大きく左右されると考えています。突然変異と放射線耐性はあまり相関がないと思います。中川：ありがとうございます。佐野：時間がだんだん迫ってきているのですが，今回のシンポジウムの環境耐性，いわゆるストレス耐性ですね。全般に通じるようなご質問のようなものがあれば，ぜひご質問願いたいと思うのですが。演者の先生方でも，その点に関して何かご発言があれば，非常に・・・。鳴海：杉本先生に質問なのですが，休眠耐性，休眠性と放射線耐性というのは関係するのでしょうか。杉本：乾燥，低温，止揚，そういったストレスに対しては，休眠するときにストレス耐性が得られるのですが，その場合，水が少ないので，その辺，多少，放射線に対する反応というのは違うかもしれないのですが，残念ながら，わたしもまだその部分はわかりません。鳴海：LEAタンパクがあると，放射線にも強いし，乾燥にも強いという話があるのですが，休眠が深い，浅いで，放射線耐性が違うのかどうかというのも興味があるところなのですけど。杉本：面白いところだとは思うのですが，何か影響はあるのかもしれないのですが，今後，その辺，関係があるのか見ていきたいと思いますが。佐野：ほかにご質問。わたしは，演者の方に，ど

なたかにお聞きしたい質問は，こういうストレス耐性をつけると，それに対してコストはかからないのかという問題が，いろんなところで大きく意見の違うものを読むのですが，それに関して何か，方向性みたいなものをご発言がありましたら，ぜひお願いをしたいのですが。品種改良のときなんて，もろにそれが，実際の問題になるかなという気もするので，どなたか，ご意見ございますか。西澤：鉄欠乏耐性の場合ですが，わたしたちが最初につくったキレート戦略の稲というのは，大麦のゲノム断片をそのまま入れているので，オウンプロモーターで動く，ですから鉄欠乏になったときだけ働くという形では，ペナルティーはないと思います。もちろんムギネ酸をたくさんつくって外に出すという意味では，コストがかかっているとは思いますが，必要がない時は作らないので，コストはかからない。₃₅Sのプロモーターでもやってみたのですが，あまりよくない。プロモーターを工夫することが重要だと思います。佐野：ほかにコメントは。時間が随分迫ってきまして，こういう新しい技術が，夢をもって発展するには，今のようにコストというか，トレードオフのような一方がよくなれば，一方が悪くなるというようなことがないに越したことはなくて，それがあれば，いろんなオールマイティーな作物ができて，世界にはいろんな欠乏症だとか，ストレスのあるところに植えていけるような夢をわれわれは持っていいのか，それはやっぱりコストがあって駄目なのかというような大きな問題は今後の重要な耐用性に関係した問題になるかと，わたし個人では思っているのです。今回のシンポジウムの個々の，最近の成果を踏まえて，今後ますます，そういった問題が深く論じられるようになればいいなということを述べまして，このシンポジウムを一応，総合討論を終えたいと思います。どうも，皆さん，ありがとうございました。演者の方々にもう一度拍手をお願いいたします。－－　佐野先生，演者の皆さま，どうもありがとうございました。閉会のあいさつということで，放射線育種場共同利用施設長の東京大学の長戸先生からお願いいたします。長戸：昨日と今日と，熱心なご討論ありがとうございました。環境耐性機構というのは，今後もま

84

すます重要な課題になってくるはずですので，このシンポジウムを ₁つの契機としてますます発展することを期待しています。では最後に，先生方

に拍手をもって，今回のシンポジウムを終わらせていただきます。どうもありがとうございました。（了）

The lecturers and the members of the Symposium Committee · Research on Plant Breeding, Indian...

Documents

Transcript of The lecturers and the members of the Symposium Committee · Research on Plant Breeding, Indian...