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, nkurata@lab.nig.ac.jp; 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)