Formation of Corymb-like Inflorescences Due to Delay in Bolting and ...

Plant Cell Physiol. 43(3): 298–306 (2002)

JSPP © 2002

Formation of Corymb-like Inflorescences Due to Delay in Bolting and Flower Development in the corymbosa2 Mutant of Arabidopsis

Mitsuhiro Suzuki, Taku Takahashi and Yoshibumi Komeda 1

Division of Biological Sciences, Graduate School of Science, Hokkaido University, N10, W8, Sapporo, 060-0810 Japan

;

Among the wild-type ecotypes of Arabidopsis thaliana

whose shape of inflorescence is categorized as raceme, the

ecotype Landsberg harboring the erecta (er) mutation shows

a corymb-like inflorescence, namely, a compact inflores-

cence with a flattened arrangement of flower buds at the tip.

The fact that the ER gene encodes a receptor-like protein

kinase implies the presence of a signaling cascade responsi-

ble for the inflorescence morphology of flowering plants. We

report here the characterization of another mutant with a

corymb-like inflorescence, named corymbosa2 (crm2), and

the isolation of the CRM2 gene. While the er mutation

causes a severe reduction in the length of pedicels, the crm2

mutation results in a significant delay in the initiation of

internode elongation and in the development of flowers,

despite having little effect on the timing of floral induction.

Consequently, the number of flower buds is apparently

increased at the tip of crm2 inflorescence. The crm2 er dou-

ble mutant shows an additive phenotype. These results sug-

gest that CRM2 and ER may act in different ways to gener-

ate wild-type inflorescence. The CRM2 gene was isolated by

positional cloning and appears to encode a polypeptide with

no significant homology to known sequences.

Key words: Arabidopsis thaliana — Bolting — Corymb-like

inflorescence — corymbosa2 — Flower development — Inflo-

rescence development.

Abbreviations: BAC, bacterial artificial chromosome; CaMV,

cauliflower mosaic virus; CAPS, cleaved amplified polymorphic

sequence; GUS, �-glucuronidase; NLS, nuclear localization signal;

ORF, open reading frame; RT-PCR, reverse transcription-PCR; SAM,

shoot apical meristem; SEM, scanning electron microscopy; SSLP,

simple sequence length polymorphism.

The nucleotide sequence reported in this paper has been submit-

ted to GenBank under accession number AF327068 (CORYMBOSA2).

Introduction

The inflorescence shoot architecture of flowering plants

can be divided into various types according to anatomical crite-

ria such as the identity of the shoot apical meristem (SAM), the

pattern of lateral flower primordia initiation, and the degree of

internode elongation. In Arabidopsis thaliana, transition of a

vegetative shoot meristem to an inflorescence meristem is

accompanied by continuous production of flowers on the flanks

of the SAM with a spiral phyllotaxy and by rapid elongation of

pedicels (the stalk of a single flower) and stem internodes. The

resulting shape of the inflorescence is referred to as raceme

(Weberling 1989). However, the molecular basis correspond-

ing to such terminology remains largely unexplored.

The SAM is a key source of cells for shoot growth. A

number of genes involved in determination of meristem iden-

tity and in regulation of meristem activity have been identified

in Arabidopsis. The TERMINAL FLOWER1 (TFL1) gene plays

a critical role in the maintenance of indeterminate inflorescence

meristems (Alvarez et al. 1992). While CLAVATA (CLV) genes

are involved in promoting the progression of meristem cells

toward organ initiation, SHOOT MERISTEMLESS (STM)

antagonistically functions in preventing meristem cells from

being incorporated into organ primordia (Barton and Poethig

1993, Clark et al. 1996, Laux and Schoof 1997). CLV1 has

been shown to encode a receptor-like kinase with an extracellu-

lar domain composed of tandem leucine-rich repeats (Clark et

al. 1997), and this suggested the exciting possibility that a cell-

cell communication of information on cell proliferation and

differentiation at shoot meristems might be mediated by recep-

tor-ligand interactions in a similar manner to growth-factor sig-

naling cascades in animal systems. Based on genetic evidence,

a small secreted peptide encoded by CLV3 has been identified

as a likely candidate for the CLV1 ligand (Fletcher et al. 1999).

Furthermore, wuschel (wus) mutations, which result in prema-

ture termination of shoot and floral meristems as with stm,

appear to be epistatic to clv mutations, suggesting that WUS

may function as a downstream target of CLV genes (Laux et al.

1996).

On the other hand, there is little genetic information on

the regulatory mechanisms of phyllotactic patterns of flower

primordia and the lengthening of pedicels and stem internodes,

both of which also contribute to formation of the gross mor-

phology of inflorescence. Mutation in the ERECTA (ER) locus,

which is known to be harbored by the wild-type Arabidopsis

ecotype Landsberg erecta (Ler), results in a compact inflores-

cence, short pedicels, and upright blunt siliques. As a conse-

quence, a bunch of flower buds at the tip of inflorescences con-

tinuously forms an umbellate flat surface referred to as

corymbose inflorescence. The ER gene codes for a receptor-

like kinase similar in overall structure to that of CLV1 (Torii et

1 Corresponding author: E-mail, [email protected]; Fax, +81-11-706-2739.

298

CORYMBOSA2 in inflorescence development 299

al. 1996). However, it is not known how the ER gene product

performs signal transduction. To understand the molecular

mechanisms regulating the inflorescence shoot architecture, the

genes involved must be identified. Toward this goal, we have

isolated a series of Arabidopsis mutants, named corymbosa

(crm), which show corymb-like inflorescences (Komeda et al.

1998). Here we describe the phenotypic characterization of the

crm2 mutant in comparison with er. We show that the corymb-

like phenotype of crm2 is manifested in a different fashion

from that of er. Cloning of the CRM2 gene revealed that it

encodes a novel protein.

Results

Overview of the corymb-like phenotype

In wild-type Columbia (Col) plants of A. thaliana, the api-

cal inflorescence meristem and flowers at early stages are cov-

ered with flowers at later stages (Fig. 1A). This is simply due

to a rapid upward growth of pedicels in unison with flower

organ development. In er mutants, the length of pedicels as

well as stem internodes is apparently reduced, and this results

in the corymb-like phenotype of the tip of the inflorescence

(Fig. 1B, F). The crm2 mutant was isolated by screening for

mutants with a similar appearance of inflorescence as that of er

mutants (Komeda et al. 1998). The flattened arrangement of

flower buds in the crm2-1 allele is shown in Fig. 1C. We

observed that, while the total number of flowers at stages 7–13

(stages defined by Smyth et al. 1990) at the tip of the inflores-

cence was normal in er alleles, it was increased in the crm2-1

allele compared to that of the wild type (Fig. 1E–G). Further-

more, in contrast to er alleles, in which the length of fully elon-

gated pedicels is about half of that in the wild type, crm2-1

appeared to have a moderate effect on the length of pedicels.

The number of floral organs was unaffected in crm2-1, as with

er alleles. These observations indicate that the corymb-like

phenotype in crm2-1 is attributable in part to the increased

number of flower buds at the tip.

To examine the relationship between CRM2 and ER in

inflorescence development, crm2 er double mutants were con-

structed. For the er mutation, the er-105 allele, which has an

Fig. 1 Morphology of inflorescences and flower buds at the tips in

wild-type (ecotype Columbia), er and crm2 plants. (A–D) Top views

of tips of wild-type (A), er-105 (B), crm2-1 (C) and crm2-1 er-105

double mutant (D) apical inflorescences. (E–G) Flower buds at stages

7–13 (stages according to Smyth et al. 1990) decapitated from wild-

type (E), er-105 (F) and crm2-1 (G) apical inflorescences. Bars =

1 mm.

Table 1 Measurements of wild-type and crm2-1 plants

a Days from seed germination are shown.b Total number of leaves and flowers visible without dissection and

using a dissecting microscope on the primary shoot was scored.c The mean length of the first ten internodes between nodes bearing a

single flower.

Mean values � SD in 20 wild-type (Col) and crm2-1 plants are shown.

Wild type crm2-1

Number of rosette leaves 9.9�0.8 10.1�0.6

Number of cauline leaves 2.7�0.5 4.4�0.5

Days to 1-cm bolting a 28.4�1.1 37.1�1.5

Days to first-flower opening a 31.5�1.9 42.2�3.3

Total number of lateral meristems at day 32 b 29.3�1.0 30.1�1.0

Length of stem internodes (mm) c 9.4�2.0 4.9�1.6

Length of flower organs (mm)

Sepal 1.9�0.1 1.9�0.1

Petal 2.7�0.3 2.6�0.2

Stamen 2.2�0.2 1.7�0.1

Carpel 2.3�0.1 2.2�0.2

Pedicel 6.9�0.6 4.1�0.3

CORYMBOSA2 in inflorescence development300

insertion of a 4-kb DNA fragment of unknown origin in the ER

gene and putatively represents a null allele (Torii et al. 1996),

was used. We did not detect a novel mutant phenotype in dou-

ble-mutant seedlings at the vegetative stage. After bolting,

crm2-1 er-105 double mutants showed an extremely exagger-

ated corymb-like phenotype at the tip of the inflorescence (Fig.

1D), suggesting that the effects of crm2 on inflorescence devel-

opment is additive to er.

The crm2 mutation delays the timing of bolting and flower

development

Under standard long-day conditions (see Materials and

Methods), the number of rosette leaves was normal in the

crm2-1 allele. On the other hand, crm2-1 produced approxi-

mately two cauline leaves more than the wild type (Table 1).

These results suggest that the crm2 mutation affects the flower-

ing time. To determine whether or not the timing of floral

induction is delayed in crm2-1, expression of the flower-meris-

tem-identity gene APETALA1 (AP1) was examined by reverse

transcription (RT)-PCR. AP1 is known to be expressed

throughout stage 1 and 2 flower primordia and in the first and

second whorls of developing flowers but not in vegetative and

inflorescence meristems (Mandel et al. 1992). We could not

Fig. 2 Comparison of the time courses of flower initiation, opening

and internode elongation in wild-type and crm2 plants. (A) RT-PCR

analysis of the expression of AP1 transcripts. Total RNA was prepared

from whole seedlings of the wild-type (WT) and crm2-1 plants grown

for indicated days (10–22) after germination, respectively, and sub-

jected to RT-PCR experiments (see Materials and Methods). As a con-

trol, the TFL1 transcript was amplified. (B) Time course of production

of flowers on the primary shoot axis. The number of flowers after

stage 13 (anthesis) was counted. The mean values (� SD) of 20 wild-

type (square) and crm2-1 (circle) plants are plotted. (C) Time course of

growth of stem internodes. The plant height was measured from the

day of 1-cm bolt. The mean values (� SD) of 20 wild-type (square) and

crm2-1 (circle) plants are plotted.

Fig. 3 Comparison of the development of primary inflorescences in

wild-type and crm2 plants. (A) A 27-day-old wild-type inflorescence.

(B) A 36-day-old crm2-1 inflorescence. (C) A 40-day-old wild-type

inflorescence. (D) A 50-day-old crm2-1 inflorescence. Arrows indicate

variation in silique length due to reduced fertility in crm2-1. Bars =

1 mm (A, B) and 5 mm (C, D).


detect clear difference between the time courses of AP1 expres-

sion in crm2-1 and wild-type plants (Fig. 2A).

On the other hand, crm2-1 started to bolt approximately

9 d later than did the wild type (Table 1). The period from ger-

mination to anthesis (stage 13) of the first flower in crm2-1 was

also much longer than that in the wild type (Table 1; Fig. 2B).

However, the production rate of stage 13 flowers by the pri-

mary inflorescence meristem in crm2-1 was higher than that in

wild-type plants (1.6 vs. 1.2 flowers per day for the first 5 d in

the wild type; Fig. 2B). These results indicate that the crm2

mutation causes accumulation of flower buds at the tip of inflo-

rescences due to a significant delay in the initiation of bolting

and in the development of floral organs but has little effect on

the timing of floral induction. Consistent with this, the total

number of visible flowers just before bolting was apparently

increased in crm2-1 (Fig. 3A, B). However, the total number of

lateral meristems including leaves and flowers was unaffected

in crm2-1 in comparison with that in the wild type of the same

day (Table 1). At day 36, when all of the flowers in crm2-1

were at young bud stages and they were still in the center of the

rosette (Fig. 3B), wild-type plants already had flowers after

anthesis, developing siliques, and elongating internodes. Fur-

thermore, the growth of stem internodes after bolting was

decelerated in crm2-1 (Fig. 2C). The final length of an individ-

ual internode was also reduced in crm2-1 (Table 1; Fig. 3C, D).

Examination of apical inflorescence meristems by scan-

ning electron microscopy (SEM) revealed that the spiral

arrangement of flower primordia at its flanks was unaffected in

crm2-1 (Fig. 4A, B). Sections of inflorescence apices revealed

that the number of flowers at early stages was increased in

crm2-1 (Fig. 4C, D), while the apical meristem in the crm2-1

allele was normal in size and shape (Fig. 4E, F).

Reduced fertility in crm2

In addition to the corymb-like phenotype, crm2-1 exhib-

ited reduced fertility (Fig. 3C, D). Although mature siliques

with wild-type length were occasionally produced, the length

of siliques and the seed set varied along the stem. Because of

the reduced fertility and/or the delayed development of flow-

ers after stage 13, these flowers were also observed in the out-

ermost whorl of a compact flower-bud cluster in the crm2 er

double mutant (Fig. 1D), instead of developing siliques in the

er single mutant. We measured the lengths of floral organs of

stage 13 flowers (Table 1). Unlike sepals, petals, and carpels,

which exhibited normal lengths in crm2-1, the average length

of crm2-1 stamens was shorter than that of wild-type stamens.

On the other hand, crm2-1 apical inflorescence meristems con-

tinued to produce flowers for a prolonged period compared to

the wild type (Fig. 2B). As a result, the crm2-1 plants finally

had more flowers than did the wild type and exhibited a final

stature comparable to or higher than that of the wild type, in

spite of a reduction in the length of each internode. These phe-

notypes may be accounted for by reduced fertility, which often

causes a prolonged proliferative activity of apical inflores-

cence meristems (Hensel et al. 1994).

Positional cloning of CRM2

The crm2 mutation segregates as a monogenic recessive

(Komeda et al. 1998). Fine mapping experiments revealed that

the CRM2 locus is located within an 80-kb region between the

markers T13K14.160-RsaI and g13838–1.4 on chromosome IV,

which we designed based on the genomic sequence data in

GenBank (see Fig. 5A). We determined DNA sequences of all

of the predicted open reading frames (ORFs) in this region by

using the crm2-1 genome and found only a 3-bp deletion in a

putative ORF in the bacterial artificial chromosome (BAC)

clone T13K14 (Fig. 5B).

Fig. 4 Microscopic observation of wild-type and crm2 inflores-

cences. (A and B) Scanning electron micrographs of wild-type (A) and

crm2-1 (B) inflorescence apex. (C and D) Cross sections of the wild-

type (C) and crm2-1 (D) inflorescence apexes. Arrows indicate flower

buds of wild-type and crm2-1 plants at the same stage (stage 6; stages

defined by Smyth et al. 1990). Note that the number of flower buds at

early stages (asterisks) is increased in crm2-1. (E and F) Longitudinal

sections of the wild-type (E) and crm2-1 (F) inflorescence apexes.

Bars = 50 �m.


To determine whether it corresponds to the CRM2 gene or

not, a 6-kb wild-type genomic DNA fragment encompassing

the ORF (Fig. 5B) was used for complementation experiments.

After transformation of wild-type plants with this fragment,

they were crossed with homozygous crm2-1 mutants. Our

results showed that all of the F2 populations derived from four

independent crosses failed to segregate for kanamycin-resist-

ant plants with the mutant phenotype (data not shown).

Sequence determination of the endogenous CRM2 gene of the

kanamycin-resistant F2 plants (see Materials and Methods)

revealed that some of them were homozygous for the crm2-1

allele and phenotypically wild type (Fig. 6A).

We further transformed wild-type plants with an antisense

construct containing a part of this ORF (Fig. 5B) fused in

reverse orientation to the cauliflower mosaic virus (CaMV)

35S RNA promoter. Five of sixteen transgenic lines displayed

phenotypes similar to the original crm2 phenotype (Fig. 6B).

Taken together, these results indicate that this ORF is the

CRM2 gene.

Analysis of the CRM2 gene

Isolation of the corresponding cDNA by RT-PCR revealed

that the CRM2 gene contains nine introns (Fig. 5B). It encodes

a polypeptide of 942 amino acids with a predicted molecular

mass of 104 kDa (Fig. 5C). The crm2-1 mutation is located at

the second exon and it causes a deletion of a lysine (K) residue

in the CRM2 gene product (Fig. 5B, C). A BLAST search using

the CRM2 polypeptide sequence revealed no homology with

Fig. 5 Identification of the CRM2 gene. (A) Mapping of the CRM2

locus. CAPS markers on chromosome IV, g13838–1.4 and PG11, are

shown at the top. Asterisks represent the position of the CRM2 locus.

Four BAC clones around the CRM2 locus are shown below. Vertical

arrows indicate positions of CAPS markers that we designed according

to polymorphisms between Columbia and Landsberg ecotypes (see

Materials and Methods). Number of recombinant chromosomes from

870 crm2-1 homozygous plants (1740 chromosomes) of the F2 prog-

eny of a cross between crm2-1 and the wild type (ecotype Landsberg)

are indicated. (B) Structure of the CRM2 gene. Exons are indicated by

black boxes. The position of a 3-bp deletion in crm2-1 is indicated by

a vertical arrow. Arrows with restriction sites represent PCR primers

used for amplification of a genomic DNA fragment for transgenic

complementation experiments. An open bar represents a restriction

fragment used for the antisense construct. (C) The deduced amino acid

sequences of the CRM2 protein. The amino acid residues derived from

translation of the RT-PCR product are shown (one-letter code). The

functional translation stop site (TGA) is marked with an asterisk. Dou-

ble underlines indicate the site of the crm2-1 mutation. A region simi-

lar to the double-stranded RNA-binding motif is underlined.

Fig. 6 Analysis of transgenic Arabidopsis plants. (A) Complementa-

tion of the crm2 phenotype with the wild-type CRM2 gene. A wild-

type genomic DNA fragment containing the CRM2 locus was obtained

by PCR (see Fig. 5) and introduced into wild-type plants. crm2-1

homozygous plants with wild-type appearance were identified in the

F2 progeny of the cross with crm2-1 plants by PCR and sequencing of

the allele. (B) A phenotype conferred by antisense expression of the

CRM2 gene. A partial genomic fragment of the CRM2 gene (see Fig.

5B) was fused to the CaMV 35S RNA promoter and introduced into

wild-type plants. Bars = 1 mm.


known proteins. However, we found that a homologous

sequence is tandemly arrayed with the CRM2 gene in the BAC

clone T13K14. The CRM2 polypeptide sequence has 64.2%

identity overall with a deduced polypeptide of its neighboring

homolog (T13K14.80; accession number T10634). The PSORT

algorithm (Nakai and Kanehisa 1992) recognized a potential

nuclear targeting sequence (KKKK, amino acid residues 68 to

71), which is converted into KKK in crm2-1. The deduced

polypeptide of T13K14.80 also contains a nuclear targeting

sequence (KRKK, amino acid residues 56 to 59) in the corre-

sponding position (KKKK) of the CRM2 protein (data not

shown). Motif analysis using the Pfam HMM database

revealed a region of the CRM2 protein between amino acids 11

and 85 that displays similarity with the double-stranded RNA-

binding motif (Fig. 5C).

Because we detected no positive signals of CRM2 expres-

sion by RNA gel blot analysis, we carried out RT-PCR to deter-

mine where the CRM2 gene is expressed. The CRM2 tran-

scripts were mainly detected in young seedlings, root tissue and

flower buds with shoot apices (Fig. 7). Similar expression pat-

terns were also observed on the T13K14.80 gene (Fig. 7).

Discussion

There is an astonishing variety of forms of flower-bearing

branching systems, which contain different modifications of

the same basic plan often observed within the same family. As

one approach toward unravelling the molecular basis for these

divergent forms of inflorescence in flowering plants, we

focused on the inflorescence morphology in A. thaliana. As

described in detail by Weberling (1989), the raceme inflores-

cence of the family Cruciferae, to which Arabidopsis belongs,

is characterized by elongation of stem internodes of its main

axis and development of pedicels in each flower. Although er

and crm2 mutant inflorescences take on the form of a raceme at

later stages of development, these continuously keep a corymb-

like flattened arrangement of flower buds at the tip. Our analy-

sis revealed that the corymb-like form in crm2 is generated by

an essentially different mechanism from that in er.

Fig. 8 illustrates how corymb-like phenotypes are mani-

fested in these mutants. Because the timing of bolting and

flower opening was normal in er-105, which represents a puta-

tive null allele (data not shown), the er phenotype is primarily

attributable to a reduction in the pedicel length. On the other

hand, the concurrent delay in flower organ development and

bolting in crm2-1 resulted in the accumulation of flower buds

at the tips of inflorescences. Although we could not detect any

differences between AP1 expression profiles in the wild type

and crm2-1, crm2-1 produced more cauline leaves than did the

wild type, suggesting that the timing of floral induction may be

only slightly delayed. These phenotypes might be interpreted,

at least in part, by the delayed cell division rate at the cellular

level. It is less likely that the rate of initiation of flower primor-

dia is increased in crm2-1, because the total number of leaves

and flowers was unchanged in young flowering plants of the

mutant compared to wild-type plants of the same day (Table 1).

However, the interpretation of the crm2 phenotype is somewhat

complicated by the fact that the rate of production of stage 13

flowers was higher in the mutant than that in the wild type (Fig.

2B). Assuming that the rate of initiation of flower primordia is

normal in crm2-1, this result suggests that the duration of

flower organ development may be gradually restored in late

flowers and, accordingly, the phenotype must become moder-

ated. To clarify this point, further analysis of the development

of individual flowers will be needed. Smyth et al. (1990)

defined 12 stages of Arabidopsis flower development from ini-

tiation until the bud opens. Although the possibility that the

duration of certain stages in crm2 may be particularly pro-

longed cannot be ruled out, we observed no obvious accumula-

tion of flower buds of specific stages in the mutant bud cluster

(Fig. 1, 4), suggesting that the crm2 mutation may affect all

stages of flower development.

Fig. 7 RT-PCR analysis of CRM2 and its neighboring homolog

T13K14.80. Total RNA was isolated from 7-day-old seedlings (Se),

root tissue (R), rosette leaves (L), stem internodes (St), flowers con-

taining shoot apical meristems (F) and siliques (Si). One �g of total

RNA was used in each reaction. The PCR products were blotted onto

nylon membranes and hybridized with each labeled probe. As a con-

trol, the TUBULIN �9 (TUB9; Snustad et al. 1992) transcript was

amplified.

Fig. 8 Schematic representation of the inflorescence shoot architec-

ture of A. thaliana. (A–C) Overviews of the tips of inflorescences in

wild-type Columbia (A), er (B) and crm2 (C) plants of the same ages

are shown. Arrowheads indicate apical inflorescence meristems. Indi-

vidual flower buds are represented by circles.


The crm2 phenotype suggests a tight continuity between

internode elongation and flower organ development in Arabi-

dopsis. However, considering the fact that growth of stem

internodes can occur without flower development in the leafy

mutant of Arabidopsis (Schultz and Haughn 1991) and that

some mutants have been identified with a defect in internode

elongation but not in flower organ development (Fridborg et al.

1999, Hanzawa et al. 2000), a causal relationship between the

delay in bolting and that in flower organ development is

unlikely. Reduction in the final length of an individual inter-

node in crm2-1 (Table 1; Fig. 3C, D) may reflect the effect of

the mutation on the elongation rate of stem internodes after

bolting (Fig. 2C). However, as a consequence of flower pro-

duction for a prolonged period, which may be related to

reduced fertility, the final stature of crm2 plants is comparable

to or higher than that of the wild type. Taken as a whole,

CRM2 is likely to be involved in the acceleration of growth of

flower organs and stem internodes. It is well known that gib-

berellins (GAs) play a critical role in many aspects of plant

development, including internode elongation and flower open-

ing, as a plant growth regulator (Pharis and King 1985).

Although crm2 plants do not exhibit pleiotropic phenotypes

caused by GA deficiency and the crm2 phenotype can not be

restored by exogenous application of GAs (data not shown),

suggesting that crm2 plants have normal levels of endogenous

GAs, the possibility cannot be excluded that CRM2 may be

involved in GA signal transduction pathways.

We cloned the CRM2 gene by a chromosome walk. Final

proof of identification of the CRM2 gene was obtained by com-

plementation of the crm2 mutant phenotype with the wild-type

gene. RT-PCR experiments revealed that the CRM2 transcripts

are present mainly in seedlings, roots and flower buds. We

could not detect any signals of the CRM2 transcript by in situ

hybridization analysis. Given the mutant phenotype, CRM2

may be expressed in shoot and root meristems, although the

crm2-1 allele showed normal growth of the root tissue (data not

shown). The crm2-1 allele contains a 3-bp deletion correspond-

ing to one amino acid that is located in a putative nuclear local-

ization signal (NLS) of the deduced protein. Expression of the

mutated transcript was detected in crm2-1 plants (data not

shown), suggesting that it may be translated. It remains to be

determined whether the CRM2 protein is indeed targeted to the

nucleus or not. If so, deletion of the NLS in crm2-1 might pre-

vent nuclear localization of the mutated CRM2. Whatever the

functional significance of the amino acid deleted in crm2-1 is,

the possibility that the mutated CRM2 protein still retains its

function to some degree and results in the observed phenotype

cannot be ruled out. A homologous sequence was found to be

present next to the CRM2 locus. This raises another possibility

that the growth of internodes and the development of flowers in

crm2-1 may be accomplished by redundant functions of this

homologous gene. Identification of additional mutant alleles of

CRM2 and its homolog will be of considerable importance in

defining the functions of these gene products during inflores-

cence development. Motif analysis of the CRM2 protein also

shows homology to the double-stranded RNA-binding motif at

the N-terminal region. Double-stranded RNA-binding proteins

are involved in diverse cellular functions and provide further

examples of post-transcriptional gene regulation by RNA-bind-

ing proteins (Burd and Dreyfuss 1994). It remains to be deter-

mined whether CRM2 binds double-stranded RNA or not.

In conclusion, our study revealed the importance of the

timing of bolting and the balance between the rate of flower

primordia initiation and that of flower opening in determining

the inflorescence morphology. Now that the CRM2 gene has

been cloned, it should provide a useful tool for further study

and genetic manipulation of the inflorescence shoot architec-

ture of other plant species.

Materials and Methods

Plant material and growth conditions

The original crm2-1 allele was identified among the M2 progeny

of fast neutron-irradiated seeds in the Columbia (Col) ecotype (Lehle

Seeds, Round Rock, TX, U.S.A.), as previously described (Komeda et

al. 1998). The mutant was backcrossed more than three times to the

wild type before analysis. For the er mutant, the er-105 allele in the

Col background (Torii et al. 1996) was used for phenotypic and

genetic analyses.

All plants were grown on rockwool bricks supplemented with a

vermiculite in a growth chamber at 22�C under illumination with

white fluorescent light. The photoperiodic cycle was 16 h light/8 h

dark of long-day conditions.

Microscopy

For light microscopy, tissues were fixed in FAA (45% [v/v] etha-

nol, 5% [v/v] formaldehyde, and 5% [v/v] acetic acid) overnight at

room temperature, dehydrated in a graded ethanol series, and embed-

ded in Technovit 7100 resin (Heraeus Kulzer GmbH, Wehrheim, Ger-

many). Sections, 5 �m thick, were cut on a microtome (Yamato-Koki,

Asaka, Japan), attached to slide glass, and stained for 30 s in a 0.1%

(w/v) toluidine blue solution. They were examined and photographed

using a microscope (Nikon, Tokyo, Japan).

For SEM, samples were fixed in FAA and dehydrated in a graded

ethanol series and isoamyl acetate. They were then critical-point-dried

with liquid CO2. The dried materials were mounted and coated with

gold-palladium in a sputter coater (Hitachi, Tokyo, Japan). Specimens

were viewed under a scanning electron microscope (JEOL, Tokyo,

Japan) with an accelerating voltage of 25 kV.

Positional cloning

The CRM2 locus was mapped on chromosome IV by segregation

analyses using cleaved amplified polymorphic sequence (CAPS) mark-

ers (Konieczny and Ausubel 1993) and simple sequence length poly-

morphism (SSLP) markers (Bell and Ecker 1994). Further fine mapping

experiments delimited the locus to BAC clones T13K14 (accession

number AL080282) and F21C20 (accession number AL080254). The

primers we designed for CAPS on these BAC clones (see Fig. 5A) and

restriction enzymes used were as follows: T13K14–10-MseI-1, 5�-

ATTCC AGCAA CATGA GTGCC-3�; T13K14–10-MseI-2, 5�-TCTGC

AGCTG CAGAT TCACG-3�; T13K14.80-HapII-1, 5�-TCTCC ATTTC

CTCTT TGACC-3�; T13K14.80-HapII-2, 5�-TAAGT AGGCT TTTCG

ACGAG-3�; T13K14.160-RsaI-1, 5�-ACATA CTCAA GAACA

TTAAG C-3�; T13K14.160-RsaI-2, 5�-ACCGG TCTTG AATTT


ATCAT CTC-3�; T13K14.180-DdeI-1, 5�-ATGGC GGCCA TGCAA

CTAAC-3�; T13K14.180-DdeI-2, 5�-TCTTC CTGTG ATCTT CCACG-

3�; F7J7.10-EcoRI-1 (accession number AL021960); 5�-TGTTA GTTAT

GCGGT GGAGG-3�; F7J7.10-EcoRI-2, 5�-AGCTG CAACT TATTCT

CCGG-3�; T6K22.80-EarI-1 (accession number AL031187), 5�-

CATGA ACTCA TTGGA ATCCC AA-3�; and T6K22.80-EarI-2, 5�-

GCCAT TGCAT ATTTG CATGT AC-3�.

Nucleotide sequences of putative open reading frames within

the T13K14 region in crm2-1 were determined by using an

ABIPRISM377 DNA Sequencer (Applied Biosystems/Perkin Elmer,

CA) and were compared to the wild-type sequences in GenBank using

the BLAST program (www.ncbi.nlm.nih.gov/BLAST/).

RNA extraction and RT-PCR analysis

Total RNA was extracted according to the SDS-phenol method

(Takahashi et al. 1992). RT-PCR was conducted by using a TaKaRa

RNA LA PCR Kit (AMV) Ver.1.1 (TaKaRa, Kyoto, Japan) with 1 �g of

total RNA. For the first-strand cDNA synthesis, an oligo (dT)-adaptor

primer was used. Primers for AP1 (Mandel et al. 1992), TFL1

(Ohshima et al. 1997), CRM2 (accession number AF327068),

T13K14.80 and TUB9 (Snustad et al. 1992) were as follows: AP1-1, 5�-

ATGGA GAATA TGGGA ACTAG-3�; AP1-2, 5�-TCATG CGGCG

GAGGA GCCA-3�; TFL1-1, 5�-ATGGA GAATA TGGGA ACTAG-3�;

TFL1-2, 5�-GACAA TCATG AAACT AGCGT-3�; CRM2-1, 5�-TATGT

TGCGG TATGT CACCC-3�; CRM2-2, 5�-TCTGG TAGAT ACTAC

TCTAG TCAAA G-3�; T13K14.80–1, 5�-TAAGT AGGCT TTTCG

ACGAG-3�; T13K14.80–2, 5�-TCTCC ATTTC CTCTT TGACC-3�;

TUB9-1, 5�-TCTTC ATATT CAAGG AGGTC AG-3�; and TUB9-2, 5�-

TTAGG CTTCT TCTTC TTCTT CG-3�. The amplification protocol for

AP1 and TFL1 was 25 cycles of 30 s at 94�C, 30 s at 50�C, and 3 min at

72�C. The PCR products were fractionated in 1.5% (w/v) agarose gels,

stained with ethidium bromide, and visualized using UV light.

RT-PCR conditions for CRM2, T13K14.80 and TUB9 were the

same as described above except for 14 cycles for CRM2, 16 cycles for

T13K14.80, and 12 cycles for TUB9. The PCR products were fraction-

ated in 0.8% (w/v) agarose gels, transferred to GeneScreen Plus (NEN

Life Science Products, Boston, MA, U.S.A.). Blots were hybridized at

42�C with probes labeled to high specific activity by random priming

with [�-32P]dCTP using BcaBEST Labeling Kit (Takara, Kyoto,

Japan), washed at 65�C twice each with 2� SSC and 1% (w/v) SDS for

20 min, and once with 0.1� SSC for 20 min.

Plant transformation

For transgenic complementation, a 6-kb fragment containing the

CRM2 genomic sequence was amplified by PCR using the wild-type

genomic DNA as a template and sequence-specific primers with incor-

porated restriction sites. LA Taq polymerase (Takara, Kyoto, Japan),

which has a high replication fidelity, was used for PCR. The amplified

DNA was cloned as a SalI-KpnI fragment into pUC18 and sequenced.

The binary Ti vector was made from pBI101 (Clontech, Palo Alto,

CA, U.S.A.) by replacing the XbaI–SacI �-glucuronidase (GUS) gene

fragment with the multicloning site of pUC18 and then introducing the

6-kb SalI–KpnI genomic fragment. The resulting construct was intro-

duced into wild-type plants (Col) by the floral dip protocol of Clough

and Bent (1998). Transformants were selected on MS medium contain-

ing 60 �g ml–1 kanamycin and subsequently crossed with crm2-1

mutant plants to see whether the mutant phenotype could be restored

by the introduced gene in the F2 generation. The F2 plants

homozygous for the crm2-1 allele were identified by PCR that specifi-

cally amplifies the endogenous CRM2 gene and the subsequent

sequence determination of PCR products.

For the antisense expression of CRM2, the GUS gene in the

binary vector pBI121 (Clontech) was replaced with an XbaI–SacI frag-

ment from the multicloning site of pUC18. Then, a 2-kb XbaI genomic

DNA fragment containing the 5� UTR of the CRM2 gene (see Fig. 5B)

was cloned in the antisense orientation with regard to the CaMV 35S

RNA promoter. The resulting construct was introduced into wild-type

plants (Col).

Acknowledgments

We thank Masashi Hashimoto for help with plant transformation

experiments and Masahiko Ozaki for help with mapping crm muta-

tions. This work was supported in part by a grant-in-aid from the Min-

istry of Education, Science and Culture of Japan.

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(Received December 10, 2001; Accepted January 4, 2002)

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