2005 rice mutants and genes related to organ development, morphogenesis and
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Plant Cell Physiol. 46(1): 48–62 (2005)
doi:10.1093/pcp/pci506, available online at www.pcp.oupjournals.org
JSPP © 2005
Mini Review
Rice Mutants and Genes Related to Organ Development, Morphogenesis and Physiological Traits
Nori Kurata 1, 4, 5, Kazumaru Miyoshi 3, Ken-Ichi Nonomura 1, 4, Yukiko Yamazaki 2, 4 and Yukihiro Ito 1, 4
1 Genetic Strains Research Center, National Institute of Genetics, Mishima, 411-8540 Japan 2 Center for Genetic Resource Information, National Institute of Genetics, Mishima, 411-8540 Japan 3 Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, 11-8657 Japan 4 School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Mishima, 411-8540 Japan
;
Recent advances in genomic studies and the sequenced
genome information have made it possible to utilize pheno-
typic mutants for characterizing relevant genes at the
molecular level and reveal their functions. Various mutants
and strains expressing phenotypic and physiological varia-
tions provide an indispensable source for functional analy-
sis of genes. In this review, we cover almost all of the rice
mutants found to date and the variant strains that are im-
portant in developmental, physiological and agronomical
studies. Mutants and genes showing defects in vegetative
organs, i.e. leaf, culm and root, inflorescence reproductive
organ and seeds with an embryo and endosperm are de-
scribed with regards to their phenotypic and molecular
characteristics. A variety of alleles detected by quantitative
trait locus analysis, such as heading date, disease/insect re-
sistance and stress tolerance, are also shown.
Keywords: Developmental mutant — Phenotype — Physio-
logical mutant — Rice organ — Trait gene.
Abbreviations: ARGM, Asian rice gall midge; bHLH, basic
helix–loop–helix; BPH, brown planthopper; CMS, cytoplasmic male
sterility; EST, expressed sequence tag; GLH, green leafhopper; GO,
gene ontology; GRH, green rice leafhopper; LRR, leucine-rich repeat;
NBS, nucleotide-binding site; NIL, near isogenic line; PMS, photope-
riod-sensitive genic male sterility; PO, plant ontology; QTL, quantita-
tive trait locus; RIL, recombinant inbred line; SAM, shoot apical
meristem; TGMS, thermosensitive genic male sterility; TO, trait ontol-
ogy; WBPH, whitebacked planthopper; WCVs, wide-compatibility
varieties.
Introduction
The discovery and isolation of useful natural mutants and
their variants in rice breeding has taken many years and consid-
erable effort. In the latter quarter of the 20th century, many
studies were conducted to generate artificial mutants by using
γ-ray irradiation or chemical mutagens, e.g. methylnitrosourea
(MNU) and ethyl methanesulfonate (EMS). These mutants
have been used primarily for genetic studies such as identifica-
tion of genes that regulate rice morphogenesis or physiological
traits, and linkage analysis for gene mapping. In 1998, the rice
genetics committee reported 571 mutant genes (Nagato and
Yoshimura 1998). To date, nearly 2,000 trait genes including
both single Mendelian loci/genes and quantitative trait loci
(QTLs) have been recorded. Even at the present status where
tens of thousands of cDNAs and expressed sequence tags
(ESTs) have been isolated as genetic codes, most trait genes
specific for rice mutants and variant/strains have not been iso-
lated. In this review, 1,698 rice genes reported to date based on
the mutant or variant phenotypes are listed. Most of these
genes are mapped on one of the 12 rice chromosomes. Classifi-
cation of the 1,698 genes into phenotypic categories is summa-
rized in Table 1. Mutant phenotypes and gene classification fit
into particular categories for rice development, as described by
Itoh et al. in this issue. In this review, genes and mutants of key
or interesting functions in relation to rice organ development
and biological/physiological reactions have been selected from
the gene list of Table 1. The genes described in this study
should be important sources for functional genetic studies to
connect gene structure with gene functions. In fact, enormous
progress in rice genome mapping and sequencing has pro-
moted map-based cloning of these trait genes during the last
decade.
In addition to the positional cloning approach, systematic
functional genomics approaches to screen mutants using inser-
tion mutagenesis have been developed (Hirochika et al. 1996,
An et al. 2003, Ito et al. 2004b, Kim et al. 2004, Sallaud et al.
2004). Several mutants and genes from Tos17-transposed lines
and T-DNA insertion lines are included in this review. Tagged
flanking sequence databases generated for most mutant popula-
tions are used in reverse genetics approaches. The MNU-
induced mutant population established by Satoh and Omura
(1981) is thought to possess a very frequent point mutation
ratio and so could also be a powerful source for identifying
mutant lines by applying reverse genetic approaches such as
the TILLING system (Till et al. 2003). The mutant strains of
both types are collected on a large scale, and their classifica-
5 Corresponding author: E-mail, [email protected]; Fax, +81-55-981-6808.
48
Rice mutants and trait genes 49
tion according to phenotype is underway; see Appendix 1 (1)
for Tos17 tagging mutants and Appendix 1 (2) for the MNU-
induced mutants.
In this review, rice trait genes important for the under-
standing of rice are documented. Detailed data including chro-
mosomal locations, trait ontology (TO)/plant ontology (PO)
and gene ontology (GO) IDs and reference for all 1,698 trait
genes are available in the Oryzabase [see Appendix 1 (3)].
Such information could be useful for comparative studies of
organ development, and for structural variation of genes among
species and genera.
Mutants and Genes in Vegetative Organ Development
In vegetative development, leaves are generated succes-
sively from flanks of the shoot apical meristem (SAM) with an
alternate phyllotaxy. Culm, crown roots and lateral roots are
also formed in this developmental phase. A large number of
mutants defective in development of various organs have been
reported in rice, and in several cases causal genes have been
cloned. Mutants and genes associated with vegetative organ
development are categorized into four groups: (i) genes specifi-
cally expressed in the SAM; (ii) leaf mutants; (ii) culm
mutants; and (iv) root mutants.
Genes specifically expressed in the SAM
In the vegetative development of rice, the SAM maintains
itself and simultaneously generates lateral organs such as
leaves and tillers. Several different families of homeobox genes
have been cloned and analysed in plants. One of them is a
KNOX class 1 homeobox gene family which is a pivotal regula-
tor of SAM formation and maintenance in plants. In rice there
are six KNOX class 1 genes, OSH1, OSH3, OSH6, OSH15,
OSH43 and OSH71, in its genome (Sentoku et al. 1999). Five
of these genes are expressed in the SAM. Their constitutive
expression results in inhibition of regeneration from the callus
by maintaining the cells in an undifferentiated state or results in
abnormal morphology of regenerated shoots mainly on the
leaves (Sentoku et al. 2000, Ito et al. 2001). Among these
genes, only the mutant of OSH15 has been analyzed (Sato et al.
1999). The osh15 mutant shows reduced plant height and is
allelic to the dwarf mutant, d6. Thus, OSH15 is involved in the
control of normal plant height in addition to SAM formation
and maintenance. Another gene family that plays a role in
SAM is the NAC gene family that shares an evolutionarily con-
served NAC domain. Expression analysis suggests diverse
functions for each member of this family (Kikuchi et al. 2000).
Leaf mutants
In leaf development, various types of mutants have been
identified and can be categorized into two large groups; a leaf
Table 1 Classification and number of mutant/variant genes
Class of genes No. of genes Class of genes No. of genes
Vegetative organ 276 Seed 236
SAM 30 Morphological traits 55
Leaf 53 • Embryo 28
Culm 141 • Endosperm 3
Root 52 • Grain shape 24
Reproductive organ 464 Physiological traits 181
Heading date 115 • Dormancy 30
Inflorescence 111 • Longevity 3
Spikelet 46 • Storage substances 80
Pollination, fertilization, fertility 238 • Shattering 28
• Fertility restoration 16 • Taste 40
• Segregation distortion 15 Tolerance, resistance 288
• Hybrid sterility, 58 Disease resistance 134
• Hybrid weakness 22 Insect resistance 72
• Male sterility 107 Stress tolerance 82
• Female sterility 3 Characters as QTLs 297
• Meiosis 12 Grain quality 47
• Other sterilities 5 Yield and productivity 134
Heterochrony 4 Plant growth activity 72
Coloration 133 Germination 11
Anthocyanin 40 Root activity 11
Chlorophyll 77 Seed sterility 22
Others 16 Total 1698
Rice mutants and trait genes50
morphology group and a leaf color group. The leaf morphol-
ogy group contains seven types of mutants: rolled leaves, drip-
ping-wet leaves, narrow leaves, drooping leaves, blade–sheath
boundary defect leaves, glabrous leaves and hairy leaves; and
QTLs controlling leaf angle, leaf length and leaf width.
Mutants of three loci have been categorized in the droop-
ing leaf subgroup. Among them, DROOPING LEAF (DL) has
been thoroughly analyzed (Nagasawa et al. 2003, Yamaguchi et
al. 2004). DL is expressed in the central region of the develop-
ing young leaf and its loss-of-function mutation results in a
defective midrib formation and drooping leaves. DL encodes a
putative transcription factor of the YABBY family. DL is a
pleiotropic gene and also specifies carpel identity during flower
development (see Organ Identity section). Detailed analyses of
other mutants remain to be carried out.
Seven lamina joint angle QTLs, two leaf length QTLs,
five leaf width QTLs and one leaf length mutant have been
identified. In a coleoptile photomorphogenesis1 (cpm1) mutant,
coleoptiles elongate more than wild-type coleoptiles under red
light or in the darkness (Biswas et al. 2003).
There are three mutants affecting leaf blade–sheath
boundary, auricleless (aul), liguleless (lg) and collarless (col).
The auricleless (aul) mutant lacks auricles and the ligule is
rudimentary, whereas liguleless (lg) lacks ligule, auricle and
leaf collar (Maekawa 1988). The collarless mutant lacks collar
(Sanchez and Khush 1998). Another two classes of mutants,
two glabrous leaf and two hairy leaf mutants were also found in
the leaf mutants. The other leaf mutant group has abnormali-
ties in leaf color including lesion mimic spotted phenotype.
Culm mutants
Culm mutants can be categorized into eight groups;
dwarfism/elongation, floating, tiller formation, tiller angle, brit-
tleness, thickness, twisted and others. The dwarfism/elonga-
tion group includes mutants impaired in gibberellin synthesis,
gibberellin signaling, brassinosteroid synthesis and brassinos-
teroid signaling.
DWARFISM/ELONGATION
Since plant height in rice is an agronomically important
trait for breeding high-yielding cultivars, numerous dwarf and
semi-dwarf mutants have been collected. Some of them have
been shown to be gibberellin- or brassinosteroid-related
mutants.
D35, SD1 and D18 genes encode gibberellin biosynthesis
enzymes which catalyze specific steps. D35 encodes ent-kau-
rene oxidase which catalyzes the early three steps of gibberel-
lin synthesis (ent-kaurene→ent-kaurenol→ent-kaurenal→ent-
kaurenoic acid) (Itoh et al. 2004). SD1, the ‘green revolution’
gene, encodes GA20 oxidase which catalyzes three steps
(GA53→GA44→GA19→GA20) (Sasaki et al. 2002). D18
encodes GA3 β-hydroxylase that catalyzes the step from GA20
to GA1, which is an active form of gibberellin (Itoh et al.
2001). Mutants lacking each of these gene activities show spe-
cific patterns in reducing internode length. These gibberellin
synthesis genes form small gene families in the rice genome,
and the members of each gene family show different expres-
sion patterns.
In addition to gibberellin synthesis genes, several gib-
berellin signaling genes have been cloned and a signaling
mechanism has been studied. These genes are, D1, GA-INSEN-
SITIVE DWARF2 (GID2) and SLENDER RICE1 (SLR1). Loss-
of-function mutants of D1 and GID2 show a dwarf phenotype,
whereas a loss-of function mutant of SLR1 resembles the phe-
notype of an exogenous application of gibberellins on plants
(Ashikari et al. 1999, Fujisawa et al. 1999, Ikeda et al. 2001).
D1 and GID2 encode positive regulators for gibberellin signal-
ing, whilst SLR1 encodes a negative regulator. GID2 is
involved in the degradation of SLR1 (Sasaki et al. 2003). GID2
encodes an F-box protein in a component of ubiquitin ligase E3
named SCFGID2.
D1 encodes an α-subunit of the trimeric G protein, which
is localized in the plasma membrane as a complex with the β-
and the γ-subunits (Ashikari et al. 1999, Fujisawa et al. 1999,
Kato et al. 2004). The d1 mutant is insensitive to low concen-
trations of gibberellins, but still shows a response to high con-
centrations of gibberellins (Ueguchi-Tanaka et al. 2000).
Two genes have been cloned which encode proteins cata-
lyzing brassinosteroid synthesis. In d2 mutants, the second
internode from the top has been shown to be shortened,
whereas elongation of the other internodes is rarely affected
(Hong et al. 2003). The d2 mutant also shows an erect leaf phe-
notype. D2 encodes brassinosteroid C-3 oxidase (also called
CYP90D2, a member of cytochrome P450), which catalyzes a
C-3 oxidation step of brassinosteroid synthesis. In the rice
genome, one homolog of D2 has been found, which is
expressed mainly in the root.
BRASSINOSTEROID-DEPENDENT1/BRASSINOSTEROID-
DEFICIENT DWARF1 (BRD1) encodes a brassinosteroid C-6
oxidase (OsDWARF), which catalyzes the C-6 oxidation step
of brassinosteroid synthesis (Hong et al. 2002, Mori et al.
2002). The brd1 mutant shows almost no internode elongation,
a reduced ratio of leaf sheath to leaf blade, and a short root.
The brd1 mutant also shows constitutive photomorphogenesis
in the dark.
In the brassinosteroid signaling pathway in rice, only one
gene has been cloned. D61 encodes a receptor-like protein
kinase with extensive sequence identity to the Arabidopsis
brassinosteroid receptor BRI1 (Yamamuro et al. 2000). This
suggests that D61 is the receptor for brassinosteroid in rice.
The d61 mutant shows less sensitivity to exogenously applied
brassinosteroid. More endogenous brassinosteroids accumulate
in the d61 mutant than the wild-type plant. The relative ratio of
leaf blade to sheath length is also smaller compared with the
wild type. The results of these phenotypic analyses are consist-
ent with the idea that D61 is the brassinosteroid receptor.
Rice mutants and trait genes 51
FLOATING
Another group of mutants are related to floating characters
or deep water tolerance. These genes exist in the floating rice
strains, and are involved in reactions mainly for extreme elon-
gation of the internodes. These genes are analyzed as QTLs and
are described in the Stress tolerance section.
TILLER FORMATION
Seven mutants with a reduced number of tillers have been
identified. Among them, MONOCULM1 (MOC1) has been
shown to be involved in tiller bud initiation (Li et al. 2003a).
The moc1 mutant shows no tillering owing to a defect in tiller
bud formation during the vegetative phase, and produces no
rachis branches in the inflorescence. MOC1 encodes a tran-
scription factor of the plant-specific GRAS family. Expression
of MOC1 starts in the presumptive region of the axillary bud
formation, and continues until maturation of the tiller buds.
BRITTLE
Six mutants with brittle culm have been identified. Brittle
mutants are easily broken by bending. Among them, BRITTLE
CULM1 (BC1) has been studied most extensively (Li et al.
2003b). The bc1 mutant has less cellulose and more lignin than
the wild-type plant. Monosaccharide composition is also
affected in the bc1 mutant. Thus, BC1 controls the mechanical
strength of plants through the cell wall composition. BC1
encodes a COBRA-like protein and is expressed mainly in
developing sclerenchyma cells and in vascular bundles.
TILLER ANGLE AND THICKNESS
Seven genes identified as mutations or QTLs are catego-
rized in the tiller angle group. However, detailed morphological
and molecular analyses of these genes have not yet been
reported. Big uppermost culm (Buc) is categorized in the thick-
ness group.
Root mutants
In contrast to the understanding of shoot development,
analysis of root development lags behind, even though a
number of mutants have been isolated which affect various
aspects of root development. These include mutants for root
number, root branching, root length, root thickness, root
weight, root pulling force and rhizome formation. The crl1
(crown rootless1) mutant forms radicle and lateral roots
normally, but is impaired in the initiation of crown root primor-
dia (Fig. 1A, B) (Inukai et al. 2001a). Thus, CRL1 specifically
regulates initiation of crown root primordia. CROWN
ROOTLESS2 (CRL2) regulates various aspects of rice develop-
ment including initiation and subsequent growth of crown root
primordia (Fig. 1C, D, Inukai et al. 2001a). The reduced root
length1 (rrl1) and reduced root length2 (rrl2) mutations cause
short roots (Inukai et al. 2001b). In the rrl1 mutant, only the
cortical cell length is significantly reduced, whilst in the rrl2
mutant, the root apical meristem is also small (Fig. 1E, G).
Mutants and Genes in Reproductive Organ Development
Heading date
Rice is a model plant for analysis of flowering of short-
day plants, and also for comparative studies of flowering with
long-day plants such as Arabidopsis. A large number of QTLs
have been identified from a cross between wild and cultivated
rice strains and between japonica and indica strains. In one
combination, detailed analyses of every QTL have been car-
ried out using near isogenic lines (NILs) (Yano et al. 2001).
Heading date1 (Hd1) encodes a zinc-finger transcription factor
closely related to the Arabidopsis flowering gene product
CONSTANS (CO), and Hd3a encodes a protein closely related
to Arabidopsis FLOWERING LOCUS T (FT) (Yano et al.
2000, Kojima et al. 2002). Under short-day conditions, Hd1
activates expression of Hd3a and induces flowering. Under
long-day conditions, Hd1 suppresses the expression of Heading
date 3a (Hd3a) and inhibits transition to a reproductive phase.
This pattern of regulation contrasts with that of Arabidopsis in
which under long-day conditions CO (Hd1) induces flowering
through expression of FT (Hd3a) (Hayama et al. 2003).
Early heading date1 (Ehd1) encodes a B-type response
regulator of a two-component signaling system and confers
early flowering, especially under short-day conditions (Doi et
al. 2004). The effect of Ehd1 is observed in the hd1 mutant
background, but in the ehd1 mutant background Hd1 functions
normally as a floral inducer under short-day conditions and
also as a floral repressor under long-day conditions. Under
short-day conditions, expression of Hd3a is higher in the Ehd1
background than in the ehd1 background. This indicates that
Ehd1 and Hd1 function independently and that the two path-
Fig. 1 Histological characterization of seminal roots of the crl1, crl2,
rrl1 and rrl2 mutants in rice. (A–C) Transverse sections of the nodes
of wild type (A), crl1 (B) and crl2 (C). In crl1, no crown root primor-
dia is initiated (B), and in crl2 development of crown roots is impaired
(C). (D–G) Longitudinal sections of seminal roots of the wild type (D
and F), rrl1 (E) and rrl2 (G). In rrl1, the cortical cell length is signifi-
cantly reduced (E), and in rrl2 the root apical meristem is small and
the root cell length is reduced (G). Photographs are kind gifts from H.
Kitano and Y. Inukai (Nagoya University).
Rice mutants and trait genes52
ways are integrated into a flowering pathway with Hd3a.
Heading date 6 (Hd6) delays flowering under long-day condi-
tions but not short-day conditions. Although Hd6 has been
shown to encode an α-(catalytic) subunit of protein kinase CK2
(Takahashi et al. 2001), interaction with other flowering genes
and alleles remains to be studied.
In addition to these excellent examples of QTL analysis,
other QTL analyses have revealed many trait genes from a
variety of strains. Several QTLs are introduced in this mini-
review; however, QTLs showing growth activities in vegeta-
tive and reproductive organs are not incorporated. They are
agronomically important traits, such as grain quality, yield and
productivity, plant growth activity, germination, root activity
and seed sterility, and are listed in the rice genes and mutants of
Oryzabase [see Appendix 1 (3)].
Inflorescence
Inflorescence and flower mutants can be categorized into
seven groups according to the developmental step at which
mutant phenotypes are observed, such as lateral branching, lat-
eral meristem identity, flower organization, organ number,
organ identity, organ development and others.
LATERAL BRANCHING
This group of mutants has defects in the formation and
elongation of lateral branches. The mutants of this group form
fewer numbers of rachis branches and spikelets or compact
panicle(s). In this group, LAX PANICLE (LAX) has been stud-
ied most extensively. In lax mutants, spikelets are formed only
on the apices of rachis branches, and no lateral spikelets are
formed (Fig. 2A, B, Komatsu et al. 2001, Komatsu et al.
2003a). LAX is necessary for initiation of lateral meristem for-
mation. LAX encodes a putative transcription factor with a
plant-specific basic helix–loop–helix (bHLH) domain and is
expressed at the boundary between the lateral meristem and
apical meristem.
In the panicle phytomer1 (pap1) mutant, the numbers of
primary branches are increased. In addition, internode length
and number of rachis are reduced, and rudimentary and empty
glumes are elongated (Takahashi et al. 1998).
LATERAL MERISTEM IDENTITY
This group of mutants fails to specify the identity of the
lateral branch meristem as the spikelet or flower meristem.
This group includes FRIZZY PANICLE (FZP). In fzp mutants,
primary rachis branch meristems develop normally, but spike-
lets are replaced by branch meristems (Fig. 2C–E, Komatsu et
al. 2003b). As a result, branch meristems are continuously gen-
erated in the inflorescence of the fzp mutant. FZP encodes a
transcriptional activator with the ERF domain, and is expressed
in a region where the rudimentary glume primordia develop.
FLOWER ORGANIZATION
This group of mutants has defects in flower organization.
A mutant flower of this group has an abnormal number or
arrangement of whorls. Although some mutants are known,
such as extra glume-1 (eg1) and extra glume-2 (eg2), their
detailed analyses remain to be carried out.
ORGAN NUMBER
This group of mutants has defects in controlling the
number of organs developed in each whorl. In these mutants,
only the number of floral organs is affected, whereas flower
organization and organ identity are normal. floral organ
number1 (fon1) and fon2 are mutants in this group. In these
mutants, the number of floral organs such as carpel, stamen and
lodicule is increased, whereas the number of glumes such as
lemma and palea is not affected (Fig. 3B, Nagasawa et al.
1996). fon1 and fon2 control organ numbers by regulating mer-
istem size or number of meristem cells. The FON1 gene
encodes a receptor-like protein kinase with a high homology to
Arabidopsis CLAVATA1 (CLV1) (Suzaki et al. 2004).
ORGAN IDENTITY
This group of mutants has defects in specification of the
identity of organs. In rice, several genes involved in organ
identification have been identified. Among them, SUPER-
WOMAN (SPW) specifies lodicules and stamens. In spw mutant
flowers, lodicules and stamens are homeotically transformed
Fig. 2 Inflorescence images of lax and fzp mutants of rice. (A) Wild-
type inflorescence. (B) Inflorescence images of lax mutants. (C) Inflo-
rescence of fzp mutant. (D) Terminal spikelet of wild-type branch. (E)
Apical region of fzp branches. Photographs are kind gifts from J. Kyo-
zuka (University of Tokyo).
Fig. 3 Flower mutants of rice. (A) Wild-type flower. (B) fon1-2
flower. (C) spw1 flower. (D) dl flower. Photographs are kind gifts from
H. Hirano and Y. Nagato (University of Tokyo).
Rice mutants and trait genes 53
into palea-like organs and carpels, respectively (Fig. 3C,
Nagasawa et al. 2003). The SPW gene has been shown to be
identical to OsMADS16, a rice MADS-box gene. SPW is
expressed in the lodicules and stamens. These analyses indi-
cate that SPW is a class B gene and the ABC model is applica-
ble, at least in part, to rice.
DROOPING LEAF (DL), which plays a role in leaf devel-
opment (see Leaf mutants section), also specifies carpel iden-
tity (Nagasawa et al. 2003, Yamaguchi et al. 2004). In the dl
mutant, the carpel has been homeotically transformed into the
stamens (Fig. 3D). In flower development, expression of DL
starts in the presumptive region where carpel primordium is
initiated, and continues in the carpel primordium. Thus, DL
specifies the identity of only a single whorl. In this respect, the
ABC model is modified in rice.
Another gene involved in specification of organ identity is
LEAFY HULL STERILE1 (LHS1) (Jeon et al. 2000). In the lhs1
mutant flower, lemma and palea show a leaf-like appearance.
Lodicules also become leafy. The number of stamens is
reduced. In some flowers, a new abnormal flower is formed in
the whorl of the stamens, indicating that specification of floral
meristem identity is incomplete. LHS1 is identical to
OsMADS1, a close member of Arabidopsis AP1. LHS pro-
motes determination of the floral meristem identity and specifi-
cation of the lemma, palea and lodicules. Other genes have
been cloned which have resulted in homeotic transformation of
floral organs when they are ectopically expressed or their anti-
sense is expressed (Kang et al. 1998, Kyozuka and Shimamoto
2002).
ORGAN DEVELOPMENT AND OTHERS
Mutants of the organ development group have defects in
development of each organ and show abnormal development of
awn, lemma and palea after specification of their organ iden-
tity. Twenty genes or QTLs involved in awn development are
known, whilst six genes are known for each of lemma and
palea. In malformed lemma1 (mls1) and mls2, morphological
abnormalities in the lemma and low pollen fertility are
observed, and in mls3, lemma and palea are malformed (Taka-
mure and Kinoshita 1992). Mutants involved in gametophyte
development are described below.
Sexual Reproduction Processes
In this section, we describe the genes and mutations asso-
ciated with gamete or seed sterility.
Male sterility and fertility restoration
Male sterility, for which a large number of mutants are
found, is classified into four major groups: male sterility
caused by cytoplasmic male sterility (CMS), photoperiod-sen-
sitive genic male sterility (PMS), thermosensitive genic male
sterility (TGMS) and other genic male sterilities. CMS, PMS
and TGMS can be used for practical hybrid production. The
CMS lines require combination with the fertility restorer lines
to maintain a hybrid system, whereas alteration of environmen-
tal conditions, such as day length and temperature shift, can
restore fertility in PMS and TGMS (Liu et al. 2001, Wang et al.
2003).
In Oryza sativa, only a single CMS system has been thor-
oughly studied using cybrids with the cytoplasm of cv. Chinsu-
rah Boro II (Boro, indica rice) and the nuclear genome of cv.
Taichung 65 (T65, japonica rice), called ms-bo type or BT type
CMS (Shinjo 1975, Shinjo 1984). A mitochondrial atp6 and a
unique sequence orf79 downstream of atp6 are known to relate
to CMS (Kadowaki et al. 1990, Iwabuchi et al. 1993, Akagi et
al. 1994). The Rf-1 gene has been reported to encode a protein
with a mitochondrial transit peptide and pentatricopeptide
repeat (PPR) (Kazama and Toriyama 2003, Komori et al.
2004). On the other hand, neither PMS nor TGMS genes have
been isolated as yet. Ku et al. (2003) reported that pro-
grammed cell death of premature tapetum is associated with
TGMS in rice.
Several other genic mutations exhibiting pollen sterility or
abnormal anther development have also been reported (mostly
designated ms). Recently, the aid1 mutant showing a defect in
anther dehiscence was identified using an Ac/Ds transposon
tagging system (Zhu et al. 2004). The AID1 gene encodes a
novel protein with a single MYB DNA-binding domain.
Hybrid sterility and reproductive barrier
Although there are several genic models for hybrid steril-
ity, only two models are applied to interspecific rice hybrids.
One is the ‘single locus sporo-gametophytic interaction’ model,
and the other is the ‘duplicate loci gametic lethal’ model (Oka
1974, Sano et al. 1979).
Gamete eliminator genes play key functions in preferen-
tial inheritance of one of the alleles in the heterozygous F1
plants, and support the former model. The Mendelian segrega-
tion of marker genes linked with the gamete eliminator is dis-
torted in progenitors. Many gamete eliminator loci, designated
as S in most cases, have been identified in various cross-combi-
nations among the Oryza species. In the cross-combination
between O. sativa and O. glaberrima, at least seven S loci have
been identified using NILs of a T65 background (Sano 1983,
Sano 1990, Doi et al. 1998, Doi et al. 1999, Taguchi et al.
1999). The loci S1, S3, S19 and S20 act to abort pollen carry-
ing the T65 haplotype, and the loci S2 and S21 act to abort
pollen carrying the glaberrima haplotype (Fig. 4A–D). Hetero-
zygous plants in the S18 locus probably exhibit a defect in male
sporocyte development. In addition to the gamete eliminator
alleles, Ikehashi and Araki (1986) have reported the existence
of a neutral S allele (S5n) in javanicas, called wide-compatibil-
ity varieties (WCVs). The WCVs enable generation of fertile
F1 plants when crossed with japonica and indica, whilst
japonica–indica hybrids exhibit high sterility and are primarily
attributed to the S5 locus. Thus the WCVs have actually con-
tributed to overcoming hybrid sterility (Wan et al. 1993). In
Rice mutants and trait genes54
addition to S genes, many loci or genes distorting Mendelian
segregation of flanking marker genes in interspecific hybrids
have been identified, many of which are designated ga.
Recently, Harushima et al. (2002) reported that many map posi-
tions for numerous segregation distorter loci of japonica and
indica strains differed with cross-combinations, suggesting
rapid evolution of reproductive barrier genes.
Only a few pairs of loci involved in complementary F2
hybrid sterility support another genic model. Recently, Kubo
and Yoshimura (1999) reported a pair of complementary loci
for F2 hybrid sterility, hsa1 on chromosome 12 and hsa2 on
chromosome 8. The double recessive plant causes female ste-
rility (Fig. 4E, F), whilst the single recessive homozygotes of
each locus exhibit moderate sterility.
Fig. 4 Hybrid sterility found in the genus Oryza. (A–D) Pollen from
cross-combination between O. sativa L. and O. glaberrima Steud.
stained with I2-KI solution. (A) O. sativa L. cv. T65. (B) O. glaber-
rima Steud. (IRGC1104038). (C) An F1 hybrid. (D) A near isogenic
line heterozygous for the S21 locus. (E and F) Paraffin sections of the
matured embryo sac in the F1 plants between cv. Asominori (japonica)
and cv. IR24 (indica). (E) Asominori (wild type). (F) A collapsed
embryo sac in the double recessive plants for the hsa1and hsa2 loci.
(G) Hybrid weakness segregated in the F2 population between O.
sativa L. and O. glumaepatula Steud. From left to right, O. sativa
(T65), a vigorous progeny, two progenies exhibiting hybrid weakness
(indicated by arrows), and O. glumaepatula (IRGC1105688). Photo-
graphs are kind gifts from K. Doi (Kyushu University) and T. Kubo
(Cancer Institute).
Fig. 5 Phenotypes of pair1 and msp1 mutants of rice. (A) Twelve
bivalents at diakinesis in the wild type. (B) Twenty-four univalents in
the pair1 mutant. (C) Normal anaphase I in the wild type. Magenta and
green represent chromosomes and tubulin fibers, respectively. (D)
Chromosome non-disjunction at anaphase I of the pair1 mutant. (E) A
normal megaspore mother cell (MMC, arrowhead) formed in an ovule
of the wild type. (F) Plural MMCs (arrowheads) in a single ovule of
the msp1 mutant. (G and H) In situ hybridization of the MSP1 mRNA
against a longitudinal section of the wild-type anther (G) and ovule
(H). (G) PMC, pollen mother cell; Ta, tapetum; Ml, middle layer; En,
endothecium; Ep, epidermis. The tapetum layer is intensely stained,
but the PMCs are not. (H) The nucellar cells are stained, but the MMC
(arrow) is not.
Rice mutants and trait genes 55
Hybrid weakness
Hybrid weakness or hybrid breakdown is not directly
related to sexual reproduction, but is worth noting in this sec-
tion because they are one of the components of the reproduc-
tive barriers. All hw loci reported so far (Hwa, hwb, Hwc, hwd
and hwe) complementarily affect viability of F1 or F
2 plants
(Oka 1957, Amemiya and Akamine 1963, Chu and Oka 1972,
Fukuoka et al. 1998, Kubo and Yoshimura 2002). The plants
affected by the complementary gene sets generally show a
small number of tillers, short culms and panicles, chlorosis or
necrosis of leaves, absence of seed sets, root growth inhibition,
and so on (Fig. 4G).
MEIOSIS
In the majority of eukaryotes, chromosome synapsis is
mediated by an evolutionarily conserved, tripartite, protein
structure called the synaptonemal complex (SC) (Wettstein et
al. 1984, Zickler and Kleckner 1999). Synaptic mutants are
classified into two categories, asynaptic and desynaptic
mutants (Li et al. 1945). The former class is characterized by
partial or complete inhibition of the synapsis of homologous
chromosomes, whereas the latter shows precocious separation
of bivalents following chromosome pairing. In O. sativa, many
asynaptic (as) and desynaptic (ds) mutants have been isolated
so far (Katayama 1963, Kitada and Omura 1983).
Recently, Nonomura et al. (Nonomura et al. 2004a, Non-
omura et al. 2004b) identified the rice meiotic genes PAIR1 and
PAIR2 from sterile mutant lines tagged with Tos17 (Hirochika
et al. 1996). The loss-of-function mutations of two PAIR genes
result in asynapsis and chromosome non-disjunction in male
and female meiocytes (Fig. 5A–D). The PAIR1 gene encodes a
novel nuclear protein (Nonomura et al. 2004a). PAIR2 encodes
a protein homologous to the yeast HOP1 and Arabidopsis
ASY1 (Nonomura, K., Eiguchi, M., Nakano, M., Suzuki, T. and
Kurata, N. unpublished data).
OTHER STERILITIES
There are only a few other genes or mutations that are
involved in sexual reproduction but not classified in the above
categories. In the msp1 mutant flowers, the number of male and
female sporocytes increases abnormally (Fig. 5F) (Nonomura
et al. 2003). The MSP1 gene encodes a putative leucine-rich
repeat (LRR) receptor-like protein kinase, and is expressed in
the nurse cells surrounding the male and female sporocytes
(Fig. 5G, H). Lee et al. (2004) identified the OsCP1 gene from
T-DNA-tagged mutant lines. The oscp1 mutant showed a sig-
nificant defect in pollen development, in addition to dwarfism.
OsCP1 expression is observed mainly in anther locules but not
in vegetative tissues. OsCP1 encodes the papain family
cysteine protease.
Kaneko et al. (2004) identified a MYB domain gene
OsGAMYB, from the Tos17-tagged lines. The osgamyb mutant
exhibits defects especially in anther and pistil development, but
not in vegetative tissues. GAMYB is a positive transcriptional
regulator of gibberellin-dependent α-amylase (Gubler et al.
1995). Even though OsGAMYB is also highly expressed in the
aleurone layer of rice seeds, one of its essential functions might
be in pollen development (Kaneko et al. 2004).
Large-scale monitoring of specific gene expression in reproduc-
tive organs
Using cDNA microarray analyses and in situ hybridiza-
tion techniques, Endo et al. (2004) revealed 259 non-redun-
dant cDNA clones specifically or predominantly expressed in
rice anthers, and classified into four groups. Lan et al. (2004)
reported the 253 ESTs exhibiting differential expression dur-
ing pollination and fertilization.
Genes and Mutants Identified as Seed Characters
Embryo and endosperm mutants are divided into catego-
ries of embryo, endosperm and grain shape for the morphologi-
cal characters, and dormancy, longevity, storage substances,
shattering and taste for the physiological characters.
Embryo
A large number of mutations affecting various aspects of
seed development have been reported in rice. Rice seeds con-
sist of two distinct parts, an embryo and an endosperm. Over
200 embryo mutants reported to date (Nagato et al. 1989,
Kitano et al. 1993, Hong et al. 1995a) are categorized into five
groups: embryoless, deletion of embryonic organs, altered
embryo size, modified organ position and aberrant morphology.
EMBRYOLESS AND ORGAN DELETION
The embryoless1 (eml1) mutant develops seeds that lack
an embryo (Fig. 6A) (Hong et al. 1995a, Hong et al. 1995b).
The eml1 embryo degenerates at the early stage (∼3 d after pol-
lination), but endosperm development seems normal. Notably,
manifestation of an embryoless phenotype depends on the
growing temperature.
The globular embryo (gle) mutants develop embryos with
a globular shape and completely lack embryonic organs (Fig.
6B). At least four loci (gle1–gle4) cause this phenotype. Analy-
sis of a gle4 embryo using molecular markers indicates that
GLE4 plays a role in radial pattern formation during rice
embryogenesis, but not in formation of embryonic organs
(Kamiya et al. 2003). The club-shaped embryo (cle) mutants
fail to form both a shoot and a radicle, as does the gle muta-
tions (Fig. 6C). Unlike the gle embryos, palisade-shaped cells
characteristic of the scutellar epithelium are formed in the
whole epidermis of cle. Since the palisade-shaped cells of cle
express an α-amylase gene, it is considered that the most cle
embryo comprises the scutellum (Hong et al. 1995a).
The organless (orl) mutants also lack both a shoot and a
radicle (Fig. 6D). Unlike cle1, the orl1 embryo differentiates
palisade-shaped cells in the epidermis-facing endosperm, sug-
gesting that embryonic polarity is not affected in orl1. Consist-
Rice mutants and trait genes56
ently, tissue specificity of OSH1 gene expression is maintained
in orl1 (Sato et al. 1996).
Four shootless (shl) mutants, shl1, shl2, shl3 and shl4,
have been reported to be indispensable in shoot formation dur-
ing embryogenesis (Satoh et al. 1999). Of these, shl1, shl2 and
shl4 exhibit indistinguishable phenotypes; complete loss of
shoots, coleoptile and epiblast and normal radicle formation
(Fig. 6E). In another mutant, shl3, in addition to deletion of the
shoot, the radicle is exogenously produced and cells in the
remaining tissues are highly vacuolated. SHL1, SHL2 and
SHL4 function upstream of OSH1 (Satoh et al. 1999). At least
two loci, RAL1 and RAL2, are required for radicle formation.
The ral embryo seems to be truncated in the apical–basal direc-
tion but develops a shoot. After germination, the ral plant pro-
duces normal crown roots. Thus, it is supposed that the ral
phenotype bears root-producing ability, but is caused by dele-
tion of the basal embryonic region where the radicle develops.
In addition, ral1 affects vascular pattern formation (Scarpella et
al. 2003).
ORGAN MORPHOGENESIS
Two shoot organization (sho) mutants, sho1 and sho2,
show severe defects in embryonic organ development. In sho
embryos, the first to the third leaves are malformed, but the
radicle develops normally (Fig. 6F). The sho1 plant produces
malformed leaves with a random phyllotaxy and a short plasto-
chron, resulting in deformed shoot architecture (Itoh et al.
2000).
EMBRYO SIZE
Two types of mutations causing either a reduction or
enlargement of embryo size have been reported (Hong et al.
1996). The giant embryo (ge) mutants have a 1.2–1.5 times
longer embryo and reduced endosperm (Fig. 6G). The scutel-
lum is enlarged but the sizes of the shoot and radicle are not
affected (Hong et al. 1996). Three reduced embryo (re)
mutants, re1, re2 and re3, exhibit a reduction in embryo size.
The re embryo is less than half the length of the wild-type
embryo, and shows a reduction in all embryonic organs includ-
ing the apical meristems and the enlarged endosperm (Fig. 6G)
(Hong et al. 1996). Anatomical and double mutant analyses
using ge, re and other mutants suggest that the primary func-
tion of GE and RE resides in endosperm development, not in
embryogenesis (Hong et al. 1996).
ORGAN POSITION
The APD1 locus is related to organ position. In the apical
displacement (apd) mutant embryo, the shoot is formed at the
apex of the embryo and the radicle at the center of the embryo
(Fig. 6H). This phenotype is due mostly to an enlargement of
the basal region of the embryo and underdevelopment of the
scutellum (Hong et al. 1995a).
Fig. 6 Phenotypes of rice embryo mutants from median longitudinal sections. (A) eml1 seed showing no embryo. (B) gle1 embryo without
organs. (C) cle1 embryo without organs. (D) orl1 without organs. (E) shl1 embryo lacking a shoot but with a normal radicle. (F) sho1 embryo
with an aberrant shoot lacking a coleoptile but with a normal radicle. (G) Wild-type embryo (left), ge embryo with enlarged scutellum (center)
and re1 embryo in which every organ is reduced (right). (H) apd1 embryo with apically displaced shoot and radicle. (I) enl large embryo lacking
endosperm. S, shoot; Sc, scutellum; R, radicle. Photographs are kind gifts from Y. Nagato (University of Tokyo).
Rice mutants and trait genes 57
Endosperm
Because the cereal endosperm is an important staple diet
in many countries, studies on genes acting in the endosperm
are important issues for both basic research and plant breeding.
Although a large number of genes that are associated with stor-
age substances in the endosperm, such as protein and starch,
have been identified, analysis of genes regulating endosperm
development is limited. Only one mutant affecting endosperm
development has been reported. The endospermless (enl)
mutant fails to form an endosperm (Fig. 6I) (Kageyama et al.
1991). After fertilization, the enl1 endosperm starts to develop
but degenerates at an early stage (∼3 d after pollination), result-
ing in the production of a very large embryo (Kageyama et al.
1991, Miyoshi et al. 2000). The mutations associated with
embryo size, ge and re, are also supposed to affect endosperm
growth (Hong et al. 1996).
SEED MATURATION
To date, a large number of rice genes related to seed matu-
ration processes, mainly starch and storage protein synthesis
and their accumulation, have been identified.
Rice seeds accumulate various kinds of substances as
nutrients to support seedling growth. Starch, which consists of
amylose and amylopectin, is the major source of nutrition for
seedling growth. The Waxy (Wx) gene encodes a granule-bound
starch synthase involved in amylose synthesis (Nelson and Pan
1995). Two naturally occurring Wx alleles, Wxa and Wxb, are
known (Sano 1984, Sano et al. 1986). Rice cultivars (mainly
japonica cultivars) with a Wxb allele produce lower amounts of
Wx protein than those (mainly indica cultivars) carrying Wxa
(Sano et al. 1986). Wxb carries a substitution mutation at the 5′
spliced site of the first intron, and causes a low content of amy-
lose (Cai et al. 1998, Isshiki et al. 1998). The dull (du) muta-
tions also affect amylose content. In du-1 and du-2, the amount
of spliced Wxb mature transcript, but not Wxa transcript, is
reduced (Isshiki et al. 2000).
Cereal seed proteins have been classified into four types,
water-soluble albumin, salt-soluble globulin, alcohol-soluble
prolamin and acidic or basic solution-soluble glutelins. Among
them, glutelin has been studied intensively because it is the
most abundant storage protein occupying 60–80% of the total
endosperm protein. Glutelin precursors are encoded by a multi-
gene family that consists of the GluA and GluB subfamilies
(Takaiwa et al. 1991). Mutations showing a reduction or lack of
a particular subunit or precursor polypeptide have been identi-
fied. Three loss-of-function mutants, gulutelin1 (glu1), glu2
and glu3, have defects in subunits 1a, 2a and 3a, respectively.
A dominant mutation of Low glutelin content1 (Lgc1) causes a
reduction in glutelin content. Unlike the above glu mutations,
the Lgc1 mutation has been found to influence the amount of
most glutelin subunits (Iida et al. 1997). It is revealed that Lgc1
suppresses accumulation of glutelin gene family transcripts via
RNA silencing (Kusaba et al. 2003).
Seed dormancy
Since seed dormancy is a complex and quantitative char-
acter influenced by both genetic and environmental factors, the
regulatory mechanism of dormancy is not well understood.
OsVP1 is a rice ortholog of VP1 in maize and ABI3 in Arabi-
dopsis (Hattori et al. 1994). Molecular analysis and in situ
expression patterns indicate the involvement of OsVP1 in seed
maturation processes including dormancy, as in maize and Ara-
bidopsis (Hattori et al. 1995, Miyoshi et al. 2002). The rice
vivipary (riv) mutants, riv1 and riv2, exhibit precocious germi-
nation before harvesting and reduced sensitivity to abscisic
acid (Miyoshi et al. 2000).
QTL analysis has detected dormancy-related genes. By
using RILs (recombinant inbred lines) between cultivated and
wild rice strains, 17 QTLs have been detected (Cai and
Morishima 2000). In addition, five QTLs have been detected
using BILs (backcross inbred lines) derived from a backcross
of Nipponbare/Kasalath/Nipponbare (Miura et al. 2002).
Heterochrony
The heterochronic genes control the temporal regulation
of development and greatly affect plant architecture. Two hete-
rochronic genes have been identified and characterized in rice.
One is PLASTOCHRON1 (PLA1) (Itoh et al. 1998). In the pla1
mutant, primary rachis branches in the panicle are converted
into vegetative shoots (Fig. 7B). Because vegetative to repro-
ductive phase transition occurs normally, the vegetative phase
is elongated and both vegetative and reproductive programs are
expressed simultaneously in the pla1 mutant. Thus, PLA1 is a
heterochronic gene controlling proper termination of the vege-
tative phase. The pla1 mutant also shows short plastochron, an
enlarged SAM, small leaves, bending of the lamina joint and
dwarfism (Fig. 7A). PLA1 encodes a member of CYP78A, a
subfamily of the large cytochrome P450 family, and is
expressed in young leaves and bracts (Fig. 7C, D, Miyoshi et
al. 2004).
The mori1 mutant reiterates the wild-type second leaf
stage and fails to induce adult phase (Asai et al. 2002). MORI1
is a heterochronic gene playing an important role in juvenile to
adult phase transition.
Tolerance and Resistance
Rice has evolved many kinds of resistance or tolerance
genes against biotic and abiotic stresses. Almost all of the
resistance or tolerance genes have been found as variant alleles
in a variety of cultivated and wild strains.
Disease resistance
A large number of resistant genes or alleles specific to
individual races of fungi and bacteria have been identified.
Two serious and well-studied rice diseases are rice blast, a fun-
gal disease caused by Magnaporthe grisea, and bacterial bright
Rice mutants and trait genes58
caused by Xanthomonas oryzae. Most resistance genes identi-
fied for bacterial and fungal pathogens have nucleotide-bind-
ing sites/LRRs (NBS-LRR) and/or serine/threonine receptor
kinase domains. In total, >20 resistance genes to Xanthomonus
are identified, and three genes are cloned; Xanthomonus resist-
ance gene 1 (Xa1), Xa21 and Xa26, encoding LRR receptor
kinase proteins (Yoshimura et al. 1998, Wang et al. 1998, Sun
et al. 2004). Among >60 blast resistance genes/alleles includ-
ing QTLs, two genes are cloned: Pyricularia oryzae (Mag-
naporthe grisea) resistance gene ta (Pi-ta) encoding NBS-LLR
and a gene found in a resistant strain Zhai Ye Qing8 (ZYQ8)
encoding a protein carrying a low homology to the serine/thre-
onine kinase domain and a calmodulin-binding domain (Wang
et al. 1999, Zheng et al. 2004).
A single base change in Pi-ta confers a difference between
resistance and susceptibility (Bryan et al. 2000). Avirulence
genes in the infectious blast fugi are also characterized. An
avilurence gene that is interactive with Pi-ta has also been iso-
lated (Orback et al. 2000). In addition, an intensive gene-for-
gene analysis has been carried out of the multiple steps of the
infectious process of Magnaporthe in rice (Sesuma and
Osbourn 2004), and a variety of cDNAs induced during the
defense steps in the rice blast-resistant mutant have also been
identified (Han et al. 2004).
Recently, an approach has been used to identify mutants
which confer breakdown of Xa21-mediated resistance from
thousands of mutant strains (Wang et al. 2004). Xa21 is a wide
spectrum resistance gene for multiple varieties of X. oryzae.
Therefore, findings of the breakdown mutants for Xa21 resist-
ance would clarify genes composed of the Xa21 multispectrum
defense pathway.
A hundred disease-resistant or defense-response gene-like
sequences have been mapped into several clusters on the rice
chromosomes, and some of them coincide with QTLs or major
resistance genes (Wang et al. 2001). The NBS family has
around 400 members in the rice genome (Monosi et al. 2004),
and the receptor-like kinase (RLK) family has about 1,200
members (Shiu et al. 2004).
Lesion mimic mutants
The lesion mimic mutants show symptom-like spots on
leaves and sometimes on the panicles. Over 11 mutants have
been identified and are classified as spotted leaves (spl) or cell
death and resistance (cdr) (Fig. 8A). Most of them are thought
to have defects in genes with roles in the disease defense path-
ways against attack from fungi, bacteria or viruses, althought a
few spl mutants do not show resistance. Some mutant genes
which produce lesions mimicking symptoms are well charac-
terized (Takahashi et al. 1999, Yamanouchi et al. 2002). Two
genes cloned from spl mutants are the heat stress transcription
factor (HSF) gene (Kawasaki et al. 1999), and a U-box/Arma-
dillo repeat protein gene involved in the ubiqitination pathway
(Zeng et al. 2004).
Insect resistance
More than 200 species of insects are associated with rice
plants as pests. Among them, the brown planthopper (BPH;
Nilaparvata lugens Stål), whitebacked planthopper (WBPH;
Sogatella furcifera Horvath), green leafhopper (GLH; Nepho-
tettix virescens Distant), green rice leafhopper (GRH; Nepho-
tettix cincticeps Uhler) and Asian rice gall midge (ARGM;
Orseolia oryzae Wood-Mason) are the worst pests for which
the major host resistance genes have been well studied. Most of
the >70 resistance genes reported so far have been identified
using RILs, NILs or detected as QTLs.
More than 13 resistance genes against BPH and six genes
against WBPH have been identified, and several BPH resist-
ance genes have been tagged on a molecular linkage map. An
example of antibiosis is shown in Fig. 8B–D. Rice ovicidal
response to WBPH is characterized by the formation of watery
lesions and production of an ovicidal substance, benzyl ben-
Fig. 7 Expression and mutant phenotype of PLA1. (A) Seedlings of
the pla1 mutant and the wild type. Due to the shortened plastochron,
the pla1 mutant produces more leaves than the wild type. (B) Panicles
of the pla1 mutant and the wild type. Primary rachis branches of pla1
are converted into vegetative shoots. (C and D) In situ hybridization of
PLA1 mRNA in wild-type leaves. (C) Expression of PLA1 at the base
of young leaves. (D) Expression of PLA1 in bracts.
Rice mutants and trait genes 59
zoate, which causes high egg mortality of WBPH (Seino et al.
1996, Suzuki et al. 1996). A gene with ovicidal activity to
WBPH, Ovc, and four ovicidal QTLs, qOVA-1–3, qOVA-4,
qOVA-5–1 and qOVA-5–2, have been identified (Yamasaki et
al. 2003). Ovc was the first gene to be identified that kills
insect eggs in plants.
Another type of antibiosis is found in resistant varieties
against GLH and GRH. The resistant plants cause delayed
growth and eventual death of infesting insects. The resistance
may be concerned with sucking inhibition after their infesta-
tion. So far, six loci for resistance to GRH, Grh1–Grh6, are
known. NILs carrying single resistance genes show weak
resistance (Grh1and Grh2) and susceptibility (Grh4). On the
other hand, NILs carrying two resistance genes, Grh2 and
Grh4, express strong resistance to GRH and GLH. The interac-
tion of Grh2 and Grh4 expresses a strong resistance against
two leafhopper species in rice.
Gm2 is a dominant gene conferring resistance to biotype 1
of ARGM (Diptera: Cecidomyiidae), the major dipteran pest.
Tissue necrosis, represented by a typical hypersensitive reac-
tion accompanied by maggot mortality, is observed within 4 d
after infestation of avirulent biotype 1 of the ARGM (Bentur
and Kalode 1996). Two other resistance genes, Gm6 and Gm7
(both may be identical), are tightly linked to Gm2 (Katiyar et
al. 2001, Sardesai et al. 2002).
Wild rice species are excellent genetic resources for resist-
ance genes and can also be used to integrate their resistance
loci into cultivated rice (Brar and Khush 1997). Some loci have
been confirmed as being transferred successfully into culti-
vated rice via homologous chromosome recombination. None
of the rice resistance genes to insects have yet been cloned.
Stress tolerance
Sensitivities to various stresses such as drought, cold (low
temperature), salt/osmotic stress, herbicides and metals vary
from strain to strain. QTL analysis using an F2 population,
RILs or NILs is the most convenient way to detect multiple
loci for tolerance and sensitivity (Nguyen et al. 2004). Instead
of identification of stress tolerance genes using genetic meth-
ods, another molecular approach to identify and isolate such
tolerance-related genes is employed (de los Reyes et al. 2003,
Dubouzet et al. 2003). Dubouzet et al. (2003) isolated five
stress responsible rice genes of the DREB/CBF transcription
factor by homolog hunting of Arabidopsis genes. The cloned
genes are expressed under cold or dehydration and high-salt
stress and then activate a number of target genes. Attempts to
generate stress-tolerant transgenic rice with stress tolerance-
related genes have also been made (Garg et al. 2002, Mukho-
padhyay et al. 2004). Recently, a more sophisticated genomic
approach has become available involving combinational analy-
sis of expression profiling using microarray analysis with seg-
regated progeny from a cross of tolerant and sensitive parent
strains (Cooper et al. 2003, Hazen et al. 2004).
Stress tolerance is tightly related to the developmental
stages and specific organs. For instance, metal ion and salinity
tolerance is expressed mainly in the root during the process of
ion absorption through water uptake. Cold tolerance is espe-
cially needed during the germinating and flowering stages. It
might be possible to identify tolerant mutants/genes in the
mutant/variant groups of these organs. Submergence tolerance
may be specifically important in rice, a character enabling sur-
vival during deep water stress. A group of wild rice strains and
landraces show submergence tolerance, which results in rapid
internode elongation. This specific character must also be regu-
lated by a genetic program for culum development. A recent
review and a QTL analysis for submergence sensitivity have
shown various aspects of this reaction (Jackson and Ram 2003,
Toojinda et al. 2003).
Coloration
Coloration is one of the most important characters of
plants, especially in relation to the chlorophyll biosynthesis
pathway. Tissue-specific coloration with anthocyanin is an
interesting phenomenon, which is partly integrated in the organ
developmental program. A large number of mutants and vari-
ants have been identified in these categories.
Anthocyanin and other colorations
More than 40 mutants and/or variants have been found to
possess purple coloration genes/alleles by modifying anthocy-
anin biosynthesis in rice. The divergent pattern of anthocyanin
coloration in specific organs, e.g. coleoptile, leaf axil, leaf
sheath, leaf blade, leaf margin, midrib, leaf apex, internode,
nodal ring, pericarp and stigma, is detected in the mutants/vari-
ants. It has been reported that these specific patterns in expres-
sion of anthocyanin are attributed to dysfunction of the key
regulator of anthocyanin biosynthesis R genes/alleles in rice. R
genes encode bHLH protein transcription factors and regulate
pigmentation in specific organs. One of the rice R loci, purple
Fig. 8 (A) Leaf phenotypes of spl2, spl4 and spl9 mutants. (B) A
watery lesion attacked by WBPH. (C) Resistance response of a
japonica cultivar Asominori with dead eggs. (D) Susceptible response
of an indica cultivar IR24 with live eggs. Photographs are kind gifts
from A. Yoshimura and H. Yasui (Kyushu University).
Rice mutants and trait genes60
leaf (PI), has a complex allele PI(w) composed of at least two
genes of the bHLH proteins (Sakamoto et al. 2001). The com-
plex nature of multiple alleles of the R loci may be involved in
a variety of organ/tissue-specific regulations of anthocyanin
biosynthesis. Sixteen mutants for coloration with substances
other than anthocyanin have been reported: Brown furrows of
hull (Bf), gold furrows of hull (gf1and2), gold hull and inter-
node (gh1-3), redpericarp and seed coat (Rd), and so on. These
mutants also show organ-specific coloration.
Chlorophyll
Chlorophyll synthesis is very important to all plants in
relation to photosynthesis. Chlorophyll and its encasing
organelle, the chloroplast, are both synthesized from chloro-
plast- and nucleus-encoded genes. Studies on chlorophyll and
chloroplast biosynthesis pathways have been extensively com-
piled for many plants. In rice, >70 chlorophyll mutants exhibit-
ing albino, chlorina, stripe, virescent, yellow-green and zebra
leaves have been recorded. Albino has no chlorophyll, com-
pletely lacks any green color and dies soon after germination.
Chlorina mutants have light green leaves and a low ability for
photosynthesis, thus growing weakly and depending on the
gene locus or allele. The stripe phenotype appears as a white
sectored leaf in the longitudinal direction, and is different in
size and number of white stripes among the loci. On the con-
trary, zebra has a white or yellow transverse banding phenotype
in the green leaves. This phenotype is highly variegated in
band size, frequency and color at every stage of plant growth.
Virescent mutants also show conditional chlorosis in the leaf to
different degrees depending on the temperature, strength and
wavelength of light. One of the virescent mutant inhibits the
translation of plastid transcripts during chloroplast differentia-
tion (Sugimoto et al. 2004). These analyses indicate there are
several components used in chlorophyll synthesis and the pho-
tosynthesis pathways. Most classes of chlorophyll mutants are
composed of >10 independent loci, suggesting that each bio-
synthesis pathway is constructed with at least 10 proteins that
are necessary to complete chlorophyll/chloroplast biogenesis.
One chrolina mutant has been revealed to encode OsCHLH, a
key enzyme in the chlorophyll branch biosynthesis pathway
(Jung et al. 2003).
For photomorphogenesis in rice, coleoptile photomorpho-
genesis 1 (cpm1) has been reported which shows impairment of
phytochrome-mediated inhibition of coleoptile growth and
impairment of anthesis (Biswas et al. 2003). However, this
mutant expresses almost the same level of phytochrome A, B
and C genes as in the wild type, suggesting that the CPM1 gene
is involved in phytochrome signal transduction that specifi-
cally leads to alterations in leaf and anther growth. This is one
of the classes of pleiotrophic mutants in which defects are
observed in leaves and other organs. A large number of other
mutant genes with different loci could provide genetic links to
the chlorophyll biosynthesis pathway.
Mutant and Variant Genes in Future Functional Genom-
ics of Rice
As shown in this mini-review, many trait genes revealed
by mutant and QTL analyses have been accumulated. In the
next decade, full genome sequence information should help to
promote the rapid growth in isolation and characterization of
these trait genes. The collection and positioning of many pieces
of genes in the developmental and physiological pathways will
decipher the many gene networks and thus determine the mor-
phological and developmental regulations for those gene net-
works. Morphological mutants play an indispensable role in the
study of rice development and of QTLs with regards to plant
growth reactions. However, individual gene characterization is
not enough to achieve such kinds of studies, but systematic
work using genomic and bioinformatic approaches is required.
Generation of a search system for knock-out mutants for all
rice genes, microarray analysis for mutant and variant strains,
gene/genome comparative studies with Arabidopsis, wild rice
and other cereal species, and an ideal analytical system for
handling large quantities of data using bioinformatic tools will
together promote functional genomic studies in the very near
future.
Appendix 1
(1) http://tos.nias.affrc.go.jp/~miyao/pub/tos17/
(2) http://www.shigen.nig.ac.jp/rice/oryzabase/nbrpStrains/kyushu-
Grc.jsp
(3) http://www.shigen.nig.ac.jp/rice/oryzabase/genes/geneClasses.jsp
Acknowledgments
We are grateful to Dr. Y. Nagato (The Univeristy of Tokyo) for
his valuable discussions and helpful comments, and also for kindly
providing photographs. We would also like to thank Drs. A.
Yoshimura, H. Yasui, K. Doi (Kyushu University), T. Kubo (Cancer
Institute, Tokyo), J. Kyozuka, H. Hirano (The University of Tokyo),
and Y. Inukai, H. Kitano, M. Matsuoka (Nagoya University) for their
useful advice and kindly providing photographs. Finally, we would
like to thank Dr. S. Iyama (National Institute of Genetics) for his huge
effort in selecting and gathering information for rice genes and
mutants for Oryzabase.
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(Received October 30, 2004; Accepted November 10, 2004)