Download EMF Genes Interact with Late-Flowering Genes to Regulate

Plant Cell Physiol. 39(4): 382-393 (1998)
JSPP © 1998
EMF Genes Interact with Late-Flowering Genes to Regulate Arabidopsis
Shoot Development
Ming-Der Haung and Chang-Hsien Yang'
Graduate Institute of Agricultural Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227 Republic of China
To investigate the genetic mechanisms regulating the
transition from vegetative to reproductive phase in Arabidopsis, double mutants between two embryonic flower
(emf) and 12 different late-flowering mutants were constructed and analyzed. Double mutants in all combinations
displayed the em/phenotypes without forming rosettes during early development; however, clear variations between
different double mutants were observed during late development, fwa significantly enhanced the vegetative property of
both emf mutants by producing a high number of sessile
leaves without any further reproductive growth in emfl
fwa double mutants. It also produced numerous leaf-like
flower structures similar to those in leafy apl double
mutant in emfl fwa double mutants. Nine late-flowering
mutants, ft, fca, Id, fd, fpa, fe, fy, fha, and fve, caused
different degrees of increase in the number of sessile leaves,
the size of inflorescence, and the number of flowers only in
weak emfl and emf2 mutant alleles background. Two lateflowering mutants, co and gi, however, had no effect on
either emfl and emf2 mutant alleles in double mutants.
Our results suggest that FWA function in distinct pathways
from both EMF genes to regulate flower competence by activating genes which specify floral meristem identity. CO
and GI negatively regulate both EMF genes, whereas the
other nine late-flowering genes may interact with EMF
genes directly or indirectly to regulate shoot maturation in
Arabidopsis.
Key words: Arabidopsis thaliana — Double mutants — embryonic flower mutant — Flower competence — Lateflowering mutants.
In Arabidopsis thaliana, the maturation of the shoot is
accompanied by many different developmental phase transitions (Hackett 1985, Napp-Zinn 1985, Poethig 1990).
These phase changes are accompanied by distinct morphological change such as the production of leaves of different
shapes and the production of floral organs (Medford et al.
1992, Schultz and Haughn 1993). The transition from vegetative rosette to reproductive inflorescence is the most
dramatic changes during Arabidopsis development and is
marked by elongation of the internodes, the appearance of
To whom correspondence should be addressed.
the cauline leaf without petiole in the basal nodes of the inflorescence, and finally the formation of the flowers. The
control of this vegetative-to-reproductive transition has
been extensively studied in Arabidopsis. All data indicate
that this transition is affected not only by environmental
conditions in which the plants are grown, such as photoperiod and temperature (Evans 1969, Vince-Prue 1983,
Halevy 1985-1989, Martinez-Zapater et al. 1994), but also
by endogenous factors such as the developmental state,
nutritional status, and hormonal balance of the plant
(McDaniel et al. 1992, Wilson et al. 1992, Bernier et al.
1993).
In Arabidopsis, many different early- and late-flowering mutants have been isolated and characterized from
which researchers have identified genes involved in either
delaying or promoting the timing of the rosette-to-inflorescence transition (Martinez-Zapater and Somerville 1990,
Goto et al. 1991, Koornneef et al. 1991, Zagotta et al. 1992,
Sung et al. 1992, Araki and Komeda 1993, Lee et al. 1993,
1994, Clarke and Dean 1994, Clarke et al. 1995, Putterill
et al. 1995, Eimert et al. 1995, Coupland 1995, Amasino
1996, Sanda and Amasino 1996). Late-flowering mutants
reported to date can be classified according to their responses to daylength or vernalization. For example, constans (co), gigantea (gi) and carbohydrate accumulation
mutantl (caml) are only slightly influenced by changing
'daylength or vernalization treatment (Koornneef et al.
1991, Lee et al. 1994, Putterill et al. 1995, Eimert et al.
1995). This suggests that the functions of these genes may
directly respond to environment conditions (MartinezZapater et al. 1994, Coupland 1995, Amasino 1996). The
fact that mutation in other late-flowering genes still responded to environment changes indicates that their functions are mainly involved in the process of maturation in
the plant itself. All genetic and physiological studies of
these late-flowering mutants suggest that there are multiple
pathways which promote the vegetative-to-reproductive
transition. In addition to these late-flowering mutants, several early-flowering mutants, early flower 1, 2, 3 (elfI, 2, 3),
terminal flower 1 (tfll), embryonic flower (emf), have
also been isolated and characterized (Zagotta et al. 1992,
Hicks et al. 1996, Shannon and Meeks-Wagner 1991, 1993,
Alvarez et al. 1992, Sung et al. 1992, Yang et al. 1995,
Coupland 1995). In contrast to late-flowering mutants,
early-flowering mutants flower early and produce fewer
rosette leaves than in v/ildtype Arabidopsis. Recent molecu-
382
Regulation of shoot development in Arabidopsis
lar and genetic analyses indicate some early- and late-flowering mutants, for example tfll, emfl, emf2, fve, co and/wa
also affect inflorescence and flower development (Shannon
and Meeks-Wagner 1991, 1993, Yang et al. 1995, MartinezZapater et al. 1995, Putterill et al. 1995, Madueno et al.
1996, Ruiz-Garcia 1997). This suggests that a complex regulatory network is involved in the regulation of developmental phase transition, and that many genes play multiple
roles in this process in Arabidopsis.
Among the early-flowering mutants, emf mutants are
the most extreme examples; they are characterized by the
absence of rosette growth, and inflorescences and flowers
develop directly from the embryo or callus (Sung et al.
1992). Two EMF loci, EMF1 and EMF2, have been identified and characterized previously (Yang et al. 1995). Double mutant analysis between emf and tfll, Ify, apl, ap2 or
ag support the hypothesis that EMF genes not only regulate
the rosette-to-inflorescence transition but also are involved
in inflorescence and flower development (Yang et al. 1995,
Chen et al. 1997). Schultz and Haughn (1993) have proposed that changing levels of COPS (Controllers of Phase
Switching) factors during shoot maturation initiate various
phase changes in Arabidopsis, and placed EMF genes at
the center of COPS. It is postulated that the level of EMF
activity decreases during shoot maturation, and the developmental phase transitions are initiated at threshold levels
of EMF activity (Yang et al. 1995). Thus, COPS or EMF activity specifies the fate of the shoot, and initiates different
developmental program which subsequently suppresses or
activates the morphogenetic genes responsible for specific
organ differentiation.
Since the mutant phenotypes are completely opposite
between emf and late-flowering mutants, late-flowering
genes have been thought to reduce COPS activity by negatively regulating EMF activity. Although genetic double
mutant analysis has indicated that emf are epistatic to two
late-flowering mutations co and gi (Yang et al. 1995), the
very important question of how EMF interact with different late-flowering genes to control shoot development
remains unclear. In order to further determine the function
and interaction of emf and late-flowering mutations in the
regulation of shoot development, we constructed and characterized double mutants between different em/alleles and
mutations from various late-flowering genes. Our results
demonstrate that two EMF genes and late-flowering genes
are interacting during all stages of plant development to
specify shoot maturation. Different late-flowering genes
function in parallel pathways with different degree of contribution, to regulate EMF activity negatively, directly or
indirectly, during shoot development.
Materials and Methods
Plant material—All late-flowering mutants lines (fwa-J, fca-
383
1, fd-1, fe-1, fha-1, fpa-1, ft-1, fve-1, fy-l, ld-1, gi-1, and co-1)
used in this study were obtained from the Arabidopsis Biological
Resource Center, Ohio State University, Columbus, OH. Two
emfl mutants, emf 1-1 and emf 1-2, and two emf2 mutants, emf2-l
and emf2-3 used in this research were isolated after EMS or gamma ray mutagenesis as described previously (Sung et al. 1992,
Yang et al. 1995).
Plant growth conditions—Seeds were sterilized and plated on
agar plates containing 1/2 MS medium (Murashige and Skoog
1962), kept at 4°C for 2 d, and then germinated in growth chambers under long-day conditions (16 h light/8 h dark) for 10 d
before being transplanted to the greenhouse. The light intensity of
the growth chambers was 150 fiE. Wild-type and homozygous
late-flowering mutant seedlings were transplanted to soil and
grown in greenhouses. The greenhouses were maintained at 22°C
with 16 h of light for long-day conditions.
Construction of double mutants—Because homozygous emf
mutants are all sterile, to construct the following double mutants:
emffwa-1, emf fca-1, emf fd-1, emf fe-1, emf fha-1, emf fpa-1,
emf ft-1, emf fve-1, emf fy-l, emf ld-1, emf co-1, and emf gi-1,
heterozygous emf plants were crossed with plants homozygous for
each of the late-flowering mutant alleles. Fj plants heterozygous
for the emf locus were self-pollinated and used to generate F 2
plants homozygous for the late-flowering alleles. Because the F2
plants were homozygous for these late-flowering mutant alleles,
all novel mutants in the F3 were double mutants.
Scanning electron microscopy—For scanning electron microscopy (SEM), 35-d old double mutant seedlings were fixed in 2%
glutaraldehyde in 25 mM sodium phosphate buffer (pH 6.8) at
4°C overnight, and dehydrated in a graded ethanol series. Specimens were critical point dried in liquid CO2. The dried materials
were mounted and coated with gold-palladium in a JBS sputtercoater (model 5150). Specimens were examined in a Topcon scanning electron microscope (model ABT-150S) with an accelerating
voltage of 15 KV.
Results
Characterization of 12 different late-flowering mutants
—Despite differences in degree of the severity, mutations in
late-flowering genes all caused a significant increase in leaf
number, and a delay in the timing of the rosette-to-inflorescence transition (Koornneef et al. 1991, Coupland 1995,
Amasino 1996) as shown in Table 1. Based on their responses to environmental stimulations, the 12 late-flowering mutants used in this study can be classified into three
groups. Mutations in CO, GI, and FHA are both photoperiod and vernalization insensitive. They are not influenced by
changing daylength or vernalization treatment. Mutations
in FWA, FT, and FD are photoperiod sensitive and vernalization insensitive. They are influenced by changing daylength but not vernalization treatment. Mutations in the
other six late-flowering genes, FCA, FY, FPA, FVE, LD,
and FE are both photoperiod and vernalization sensitive.
They are influenced by changing daylength and vernalization treatment (Koornneef et al. 1991, Coupland 1995). All
late-flowering mutants are recessive, except fwa, which is
dominant, and co, which is codominant (Koornneef et al.
1991).
Regulation of shoot development in Arabidopsis
384
Table 1 Effect of emfl-1 mutant allele on flowering behavior, and inflorescence and flower structures of late-flowering
mutants in Arabidopsis grown under long-day conditions
Plant
genotype
Number
Rosette
%
leaf
of
flowering number
plants"
Sessile
Number of Number
Internode
leaf
of
secondary
elongation
number*
branches' flowers'*
Flower structure
Sepal
Petal Stamen
Carpel
Fertility
0
>50% 10.2 ±1.6
0
>50% 38.9+5.7
4.5 ±0.41
0
>50%
yes
yes
no
>35
2.2 ±0.4
yes
8.5 ±1.5
>35
yes
0
2.3 ±0.52 yes
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
0
0
8.11 ±1.27
6.29±0.76
no
yes
0
0
no
3.86±0.69 15.3+6.63 sepal-carpel
no
no
no
no
no
sepal-carpel
no
no
30%
30%
0
0
6.38±1.19
5.63+0.81
yes
yes
1.93 ±0.73 7.87+1.88 yes
1.81+0.91 4.71 + 1.99 yes
no
no
yes
yes
yes
yes
no
no
58
165
106
60
25%
10%
18%
15%
0
0
0
0
5.4 ±0.52
6.83 ±1.83
6.0 ±0.53
4.25 ±0.5
no
no
no
no
0
0
0
0
4.2 ±1.93
4.33 ±1.97
3.63 + 1.06
3.25+0.5
yes
yes
yes
yes
no
no
no
no
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
emfl-1 fpa-1
emfl-1 fha-1
128
69
>50%
>50%
0
0
6.67±1.11
6.0 ±1.0
no
no
0
0
4.13±1.13 yes
4.69 ±1.75 yes
no
no
yes
yes
yes
yes
no
no
emfl-1 co-1
emfl-1 gi-1
67
53
>50%
>50%
0
0
4.2 ±0.42
5.57+1.04
no
no
0
0
2.86+0.69 yes
1.83+0.42 yes
no
no
yes
yes
yes
yes
no
no
WT (Col)
ld-1
emfl-1
20
20
75
emfl-1 fwa-1
emfl-1 ft-1
32
52
0%
5%
emfl-1 ld-1
emfl-1 fca-1
56
118
emfl-1 fd-1
emfl-1 fve-1
emfl-lfe-1
emfl-1 fy-1
" The total number of plants scored.
* The average number of sessile leaves (small leaves without petiole) for plants at the time of floral bud emergence.
c
Branches subtended by cauline leaves.
d
The average number of flowers or flower-like lateral organs for plants.
Different late-flowering mutations cause different degrees of effect on emfl-1 mutants in double mutants—
emfl-1, a weak emfl allele, usually produced a small inflorescence shoot that contained 4-5 sessile leaves and
1-3 flowers with incomplete floral organ development, as
shown in Fig. 1A (Sung et al. 1992, Yang et al. 1995). The
characterization of double mutants is based on the percentage of plant flowering, the number of leaves produced, and
the appearance of the inflorescence, branches, and the
flower organs. Double mutants in all 12 combinations
displayed the emfl-1 phenotypes during early development, for example, small shoot size, sessile cotyledons and
leaves, short hypocotyl, and oval-shaped cotyledons. However, except emfl-1 co-1 and emfl-1 gi-1 double mutants
which are indistinguishable from their respective emfl-1
single mutant parent, clear variations between different
double mutants were observed among other 10 combinations during late development. The phenotypes of double
mutants are grouped and described below based on their
similarity, summarized in Table 1, and illustrated in Fig. 1,
2, and 3.
emfl-1 fwa-1 double mutant—Double mutants between
emfl-1 and fwa-1 clearly showed an additive phenotype
(Fig. 2B, 3A) from their respective late-flowering and emfl1 parents by producing significantly more sessile leaves
(8.11) (normally only 2 to 4 in emfl-1 single mutants) with-
out further formation of any inflorescence or flower organs
45 d after germination. At this time, most double mutants
were senescent, and many small sessile leaves ceased
growth and occupied the top of the double-mutant plants
(Fig. 3A). There were no internode elongations between
sessile leaves. The result indicates that the early appearance
of inflorescence and flower structures in emfl-1 mutants
was greatly influenced by the presence of the late-flowering
mutant allele fwa-1.
emfl-1 ft-1 double mutant—The majority of the emfl-1 ft1 double mutants were not flowering, and produced a
higher number of sessile leaves (similar to those in emfl-1
fwa-1 double mutants) than in emfl-1 single mutants
before senescence. Among those double mutants which
bolted (5%), the bolting time was later, the number of
sessile leaves was higher (6.29), and there was a larger inflorescence than in emfl-1 single mutant, in which many secondary branches (3.86) and many novel lateral organs
(15.3) were produced (Fig. 2C, D). The size of the elongated inflorescence was more than five times bigger than it was
in emfl-1 single mutants, resulting in a much larger plant
(Fig. 2C). The novel lateral organs produced in the double mutant were leaf or sepal-carpel intermediate structures, bearing stigmatic papillae and ovule-like structures
(Fig. 2D, 3B). No petals and stamens were observed. Pistillike structures occasionally appeared from the axil of the
Regulation of shoot development in Arabidopsis
Wild-type
organs produced in emfl-1 ft-1 double mutants, the flowers
produced in this group were morphologically similar to
those in the emfl-1 single mutant, which contains sepals,
stamens, and a pistil. The number of the flowers, however,
was increased from 2.3 in the emfl-1 single mutant to 7.87
in emfl-1 ld-1, and 4.71 in emfl-1 fca-1 (Table 1). Therefore, the size of the double mutant plants was also larger
than those of the emfl-1 single mutant plants. The result indicates that ld-1 and fca-1 enhance both inflorescence and
flower development in emfl-1 mutants in a similar manner.
Late-flowering mutant
emfl-1 fd-l, emfl-1 fve-1, emfl-1 fe-l, and emfl-1 fy-1
emfl-1
emfl-1 fwa-l
emfll
ft-1
em/1-1 U-l
emfl-1 fail
emfl-1
emfl-1
emfl-l
emfll
emfl-1
emfl-1
fie-1
fy-1
fd-l
fe-l
fha-1
fpa-1
emfl-1 co I
emfl-1 gi-1
1
•>
*
emfl-3
emp-l
i
emf2-3 fwa-l
*•
emfl-3 ft-1
emfl-3
emfl-3
emfl-3
emfl-3
emfl-3
emfl-3
U-l
fca-1
fd-l
fve-1
fe-l
fha-1
emfl-3
emfl-3
emfl-3
emfl-3
fy-1
fpa-1
CO -1
gi-1
emfll
emfl-1
emfl-1
emfl-1
fl-l
U-l
fca-1
fd-l
emfl-1
emfl-l
emfl-1
emfl-l
emfl-1
emfll
emfl-l
fve-1
fyl
fe-l
fhafpa-1
co-I
gi-1
Fig. 1 Diagrammatic representation of morphology of the
lateral organs on Arabidopsis single and double mutants grown
under long-day conditions. Symbols are: Cl?=rosette leaf,
#=cauline leaf or sessile leaf, —-=inflorescence shoot, O =
flower, © = sepal-carpel intermediate organ, •=leaf-like organ.
novel lateral organs. The lateral organs produced in emfl-1
ft-1 double mutants were very similar to those in the leafy
apl double mutant, in which only leaves or carpelloidleaves were produced (Schultz and Haughn 1993). The
result indicates that the ft-1 mutant allele not only delays
the appearance of the inflorescence, but also severely alters
the flower formation in emfl-1 mutants.
emfl-1 ld-1 and emfl-1 fca-1 double mutants—The majority of the double mutants in this group also showed a reduction of plants bolting to 30% (Table 1). Among those double mutants which bolted (Fig. 2E, F), the bolting time and
the number of sessile leaves produced (6.38 in emfl-1 ld-1,
and 5.63 in emfl-1 fca-1, respectively) were similar to those
in emfl-1 ft-1 double mutants. The inflorescences produced in double mutants of this group were also larger than
those in emfl-1 single mutant; however, they were apparently different from those in emfl-1 ft-1 double mutants. The inflorescence also contained secondary branches (1.93 in emfl-1 ld-1 and 1.81 in emfl-1 fca-1) which
were subtended by a single sessile leaf. Unlike the lateral
double mutants—The overall phenotype of double mutants
in this group, as shown in Fig. 2G, H, were similar to those
of the emfl-1 ld-1 and emfl-1 fca-1 double mutants (Table
1). In contrast to the emfl-1 ld-1 and emfl-1 fca-1 double
mutants, no clear internode elongation and no secondary
branches subtended by a single sessile leaf were produced
in the inflorescence in this group, and plant size was
relatively smaller than that of previous groups. Double
mutants produced flowers phenotypically similar to those
in emfl-1 single mutant; the number of the flowers, however, was about twice of that of the emfl-1 single mutant
(Table 1 and Fig. 2G, H). The additive effects in this group
were less severe than those of the first three groups.
emfl-1 fpa-1 and emfl-1 fha-1 double mutants—The overall phenotype, the flowering behavior, and the structures of inflorescence and flower of the double mutants in
this group, as shown in Fig. 21, were very similar to those in
the last group, as described above (Table 1). The only difference was that majority of the double mutants in this group
bolted (>50%), compared to low percentage of double
mutants in the last group which bolted. The result indicates
that late-flowering mutant genes in this group also enhance
inflorescence development in emfl-1 mutants. The additive
effects, however, were less severe than those in the first four
groups.
emfl-1 co-1 and emfl-1 gi-1 double mutants—Unlike the
double mutants characterized in the first five groups, the
phenotype of the double mutants in this group is indistinguishable from their respective emfl-1 single mutant
parent in the number of sessile leaves produced, the size
of inflorescence, and the number of flowers produced
(Fig. 2J). The results indicate that emfl-1 is completely
epistatic to co-1 and gi-1.
Different late-flowering mutations cause similar effect
on emfl-2 mutants in double mutants—emfl-2, the most
severe emfl mutant allele, produced only carpelloid structures which were capped with stigma-like tissues; there
were never any leaves, petals, or stamens, as shown in
Fig. 2K (Yang et al. 1995). The characterization of double
mutants between 12 different late-flowering mutants and
the emfl-2 mutant was mainly based on the number and
degree of appearance of carpelloid structures. Double
mutants in all combinations except emfl-2 fwa-l displayed
Regulation of shoot development in Arabidopsis
Fig. 2 Phenotypic comparison of emfl single and emfl late-flowering double mutants. (A) emfl-1, 17 d after sowing on germination
media. Bar=0.67 mm. (B) emfl-1 fwa-1 double mutant, 40 d after sowing. Bar=0.67 mm. (C) emfl-1 ft-1 double mutant, 40 d after
sowing. Bar = 1.55 mm. (D) The lateral organs composed of sepal-carpel intermediate structures produced in emfl-1 ft-1 double mutant.
Bar=0.2mm. (E) emfl-1 ld-1 double mutant, 40 d after sowing. Bar=0.96mm. (F) emfl-1 fca-1 double mutant, 40 d after sowing.
Bar=1.2mm. (G) emfl-1 fd-1 double mutant, 40d after sowing. Bar=1.0mm. (H) emfl-1 fve-1 double mutant, 40d after sowing.
Bar=0.67 mm. (I) emfl-1 fpa-1 double mutant, 40 d after sowing. Bar=0.74mm. (J) emfl-1 gi-1 double mutant, 20 d after sowing.
Bar=0.67 mm. (K) emfl-2, 30 d after sowing. Bar=0.46 mm. (L) emfl-2 fwa-1 double mutant, 30 d after sowing. Bar=0.46 mm.
Regulation of shoot development in Arabidopsis
387
Fig. 3 Scanning electron micrographs of inflorescence and flower structures of various emffwa and emfft double mutants. (A) Many
small sessile leaves ceased growth and occupied the top of the emfl-1 fwa-1 double mutant. Bar=235 ftm. (B) The lateral organ composed of sepal-carpel intermediate structures bearing stigmatic papillae and ovule-like structures (arrowhead) produced in the emfl-1 ft1 double mutant. Bar=116//m. (C) Numerous callus-like structures without any clear organ formation produced in the emj"1-2 fwa-1
double mutant. Arrowhead indicates the hypocotyl. Bar= 189 fim. (D) A cluster of flower-like structures composed of many sepal-petal
intermediate structures produced in the emf2-3ft-l double mutant; stigmatic papillae were occasionally formed in this sepal-petal intermediate structure (arrowhead). Bar=429^m. (E) A pistil-like lateral organ with an unfused hole (arrowhead) produced in the em/2-/
fwa-1 double mutant. Bar=209 urn.
the emfl-2 phenotypes, for example, small shoot size,
sessile cotyledons etc. Although the reduction of carpelloid
structures to sessile-leaf-like structures was observed in
some double mutants during late development; however,
variations between different double mutants were not ob-
vious. In contrast to other double mutant combinations,
emfl-2 fwa-1 showed a clearly additive phenotype. Instead
of producing carpelloid structures, numerous callus-like
structures were formed in double mutants just 20 d after
germination (Fig. 2L). These callus-like structures contin-
388
Regulation of shoot development in Arabidopsis
Regulation of shoot development in Arabidopsis
389
Table 2 Effect of emf2-3 mutant alleles on flowering behavior, and inflorescence and flower structures of late-flowering
mutants in Arabidopsis grown under long-day conditions
Plant
r iani
genotype
Number
Rosette
leaf
plants" lowering number
of
0/
/Q
Sessile •, ntsmrtfi Number of Number
J
nucruuu1 secondary
leaf
of
number' longatio n branches' flowers''
WT (Col)
ld-1
em/2-3
20
20
55
>50% 10.211.6
0
>50% 38.915.7
0
0
4.3810.63
>50%
em/2-3 fwa-1
em/2-3 ft-1
127
26
>50%
>50%
0
0
em/2-3 ld-1
em/2-3 fd-1
emf2-3fca-l
emf2-3fve-l
emf2-3fe-l
emf2-3fha-l
78
42
79
75
103
42
>50%
>50%
>50%
>50%
>50%
>50%
emf2-3fy-l
emf2-3fpa-l
em/2-3 co-1
emfl-3 gi-1
69
27
192
30
>50%
>50%
>50%
>50%
Flower structure
Fertility
Sepal
Petal Stamen
Carpel
yes
yes
yes
>35
2.2 ±0.4
>35
8.5 ±1.5
0.3 ±0.63 2.1610.8
yes
yes
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
no
7.6 ±0.12
5.3 ±1.06
yes
yes
3.33+0.88 4.9110.79
2.55 ±0.52 8.7313.88
sepal-leaf
sepal-petal
no
no
no
yes
carpel-like
yes
no
no
0
0
0
0
0
0
8.15+0.77
4.14+0.36
4.9 ±0.89
5.73+0.9
4.2 ±0.43
6.35+0.93
yes
yes
yes
yes
yes
yes
1.21 ±0.58
0.86±0.36
1.81 ±0.91
0.1810.4
0.5710.51
0.5610.23
yes
yes
yes
yes
yes
yes
yes
no
no
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
no
no
0
0
0
0
4.21 ±0.52
4.0 ±0.0
3.5 ±0.53
3.9 ±0.88
yes
no
no
no
0.2810.47 1.9310.83 yes
1.3810.52 yes
0
0
1.5310.63 yes
0
1.7 10.48 yes
yes
no
no
no
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
3.6411.0
3.3611.15
2.5110.65
2.4510.68
2.2110.8
2.64+0.63
" The total number of plants scored.
6
The average number of sessile leaves (small leaves without petiole) for plants at the time of floral bud emergence.
c
Branches subtended by cauline leaves.
d
The average number of flowers or flower-like lateral organs for plants.
ued to grow, resulting in a large plant, without any clear
organ formation during late development (Fig. 3C). This
callus-like structure indeed was very similar to those observed in emfl em/2 double mutant described previously
(Yang et al. 1995). The result indicates that the appearance
of carpelloid-like structures in emfl-2 mutants was influenced by the presence of the late-flowering mutant allele/wa1.
Different late-flowering mutations cause different degrees of effect on emf2-3 mutants in double mutants—
emf2-3, a relatively weak emf2 mutant allele, produces a
larger inflorescence shoot than the weak emf2-l mutant.
Secondary branches subtended by a sessile leaf are occasionally observed in emf2-3 mutant before giving rise to 23 flowers with incomplete floral organ development, as
shown in Fig. 4A (Yang et al. 1995). Double mutants in all
12 combinations displayed the emf2-3 phenotypes during
early development; however, clear variations between
different double mutants were observed during late development. The phenotypes of double mutants are grouped and
described below based on their similarity, summarized in
Table 2, and illustrated in Fig. 1,3,4.
emf2-3 fwa-1 double mutant—Double mutants between
emf2-3 and fwa-1 clearly showed an additive phenotype
from their respective late-flowering and emf2-3 parents, as
shown in Fig. 4B. Unlike the emfl-l fwa-1 double mutants
which never bolted, the majority of the emf2-3 fwa-1 double mutants showed internode elongation after producing
a higher number of sessile leaves (7.6) than in emf2-3
single mutants. During late development, many novel lateral organs were formed in the elongated inflorescence
(Fig. 4B). These lateral organs were flower-like shoots corn-
Fig. 4 Phenotypic comparison of em/2 single and em/2 late-flowering double mutants. (A) em/2-3, 20 d after sowing. Bar= 1.0 mm.
(B) em/2-3/wa-1 double mutant, 40 d after sowing. Bar= 1.2 mm. (C) Lateral organs composed of many leaf-sepal intermediate structures in the em/2-3 /wa-1 double mutant. A pistil-like structure which contained several exposed ovules (arrowhead) is shown in the
center. Bar=0.15 mm. (D) em/2-3/t-1 double mutant, 40 d after sowing. Bar=1.16mm. (E) em/2-3 ld-1 double mutant, 40 d after sowing. Bar=1.62mm. (F) em/2-3/ve-1 double mutant, 40 d after sowing. Bar=1.2mm. (G) em/2-3/e-1 double mutant, 40 d after sowing.
Bar=0.8mm. (H) em/2-3 /y-1 double mutant, 4Od after sowing. Bar = 1.12mm. (I) em/2-3 gi-1 double mutant, 20d after sowing.
Bar=1.0mm. (J) em/2-1 , 20d after sowing. Bar=0.8mm. (K) em/2-1 /wa-1 double mutant, 40d after sowing. Bar=1.0mm. (L)
em/2-1 /t-1 double mutant, 40 d after sowing. An unfused pistil structure (arrowhead) was observed in em/2-1 /t-1 double mutants Bar=
1.25 mm. (M) em/2-1 /d-1 double mutant, 40 d after sowing. Bar =1.07 mm. (N) em/2-1 /pa-1 double mutant, 40 d after sowing. Bar=
0.73 mm. (O) em/2-1 gi-1 double mutant, 20 d after sowing. Bar=0.7 mm.
390
Regulation of shoot development in Arabidopsis
posed of many sepal-leaf intermediate structures separated emf2-3 single mutant parent in the time of bolting, the numby no internode elongation. Sometimes sepal-leaf interme- ber of sessile leaves produced, the size of the inflorescence,
diate structures were fused to form pistil-like structures and the number of flower produced (Fig. 4H, I and Table
which contained several exposed ovules (Fig. 4C). Stigmat- 2). These results indicate that emf2-3 is epistatic to four
ic papillae were occasionally observed in these sepal-leaf in- late-flowering mutants characterized in this group.
termediate structures, but the number was significantly
Different late-flowering mutations cause different dereduced. Petals and stamens were absent from these flower- grees of effect on emf2-l mutants in double mutants—
like structures. The lateral organs produced in emf2-3 fwa- emf2-l, a relatively strong emf2 allele, produces a short in1 double mutants were similar to those in leafy apl tfll tri- florescence shoot that has 5-6 sessile leaves on the stem and
ple or Ify ag double mutants described elsewhere (Schultz 1-2 flowers, as shown in Fig.4J. Each flower has sepals,
and Haughn 1993). The result indicates that the fwa-1 mu- stamens which are male-sterile, a prominent pistil, and
tant allele not only delays the appearance of the inflores- usually no petal (Yang et al. 1995). Double mutants in all
cence, but also severely alters the flower formation in emf212 combinations also displayed the emf2-l phenotypes dur3 mutants.
ing early development; however, clear variations between
emf2-3 ft-1 double mutant—Double mutants between different double mutants were observed during late developemf2-3 and ft-1 represented the second type of double mu- ment. The phenotypes of double mutants are grouped and
tant phenotype, as shown in Fig. 4D. A majority of the dou- described below based on their similarity, summarized in
ble mutants bolted after producing a slightly higher num- Table 3, and illustrated in Fig. 1, 3, 4.
ber of sessile leaves (5.3) than in the emf2-3 single mutant. emf2-l fwa-1 double mutant—Double mutants between
Double mutants also produced a large inflorescence similar emf2-l and fwa-1 clearly showed an additive phenotype, as
to that in emf 1-1 ft-1 double mutants. However, unlike the shown in Fig. 4K. The majority of double mutants showed
lateral organs which were sepal-carpel intermediate struc- inflorescence development after producing a higher numtures in emf 1-1 ft-1 double mutants, the flower-like struc- ber of sessile leaves (9.67) than emf2-l single mutants.
tures formed in the emf2-3 ft-1 double mutant were com- These were morphologically similar to those in emf2-3 fwaposed of many sepal-petal intermediate structures, and 1 double mutants. However, the inflorescence in emf2-l
stigmatic papillae were occasionally formed in these sepal- fwa-1 double mutants did not elongate very much and was
petal intermediate structures (Fig. 3D). Stamen-like struc- much shorter and more compact than in emf2-3 fwa-1 doutures were rarely observed, and pistil-like structures normal- ble mutants. emf2-l fwa-1 double mutants also produced
ly appeared from the center of these flower-like structures novel flowers similar to those in emf2-3 fwa-1 double
(Fig. 3D). The number of these flower-like structures was mutants, which were composed of only leaf- or sepal-like
significantly higher (8.73) than in the emf2-3 single mutant structures (Fig.4K). Sometimes leaf-sepal intermediate
(2.16), and many of these flower-like structures were structures were also fused to form pistil-like structures
clustered together without internode elongation (Table 2, (Fig. 4K, 3E). Stigmatic papillae were occasionally oband Fig. 3D). The result indicates that the ft-1 mutant allele served in these leaf-sepal intermediate structures, but the
greatly influences inflorescence and flower development in number was significantly reduced. Petals and stamens were
absent from these flower-like structures.
emf2-3 mutants.
emf2-3 ld-1, emf2-3fca-l, emf2-3fd-l, emf2-3fve-l, emf23 fe-1, and emf2-3 fha-1 double mutants—Similar to the
emf2-3 ft-1 double mutant, the majority of the double
mutants in this group bolted after producing a slightly
higher number of sessile leaves than the emf2-3 single mutant (Fig. 4E, F, G and Table 2). Unlike the large inflorescence produced in the emf2-3 ft-1 double mutant, double
mutants in this group only produced a slightly larger inflorescence with slightly more flowers than in the emf2-3
single mutant (Fig. 4E, F, G and Table 2). The flower structures were similar to those in the emf2-3 single mutant.
These results indicate that late-flowering mutations in this
group slightly enhance inflorescence development in emf23 mutants.
emf2-3 fy-1, emf2-3 fpa-1, emf2-3 co-1 and emf2-3 gi-1
double mutants—Unlike the double mutants characterized
in the first three groups, the phenotype of double mutants
in this group is indistinguishable from their respective
emf2-lft-l, emf2-l ld-1, emf2-lfca-l and emf2-lfd-l dou-
ble mutants—The majority of the double mutants in this
group bolted. Double mutants in this group only produced
a slightly larger inflorescence and a few more flowers than
the emf2-l single mutant (Fig.4L, M, and Table 3). Although unfused abnormal pistil structures were frequently
observed in emf2-l ft-1 double mutants (Fig. 4L), flower
structures in the other three double mutant combinations
in this group were morphologically similar to emf2-l single
mutants (Fig.4M). The results indicate that late-flowering
mutations in this group slightly enhance inflorescence development in emf2-l mutants.
em/2-7 fve-1, emf2-l fe-1, emf2-l fha-1, emf2-l fy-1,
emf2-l fpa-1, emf2-l co-1 and emf2-l gi-1 double mutants
—As shown in Fig. 4N, O and Table 3, the phenotype of
double mutants in this group is indistinguishable from
their respective em/2-7 single mutant parent in the time of
bolting, the number of sessile leaves produced, the size of
Regulation of shoot development in Arabidopsis
391
Table 3 Effect of emf2-l mutant alleles on flowering behavior, and inflorescence and flower structures of late-flowering
mutants in Arabidopsis grown under long-day conditions
Dlnrtf
riant
genotype
Number
Rosette
0/
/o
of
leaf
plants" flowering number
Sessile
Number of
Internode secondary
leaf
number* elongation branches'
ld-1
em/2-1
20
20
90
>50% 10.2±1.6
0
>50% 38.9±5.7
0
5.0 ±0.41
>50%
0
emfl-l fwa-I
47
>50%
0
emf2-lft-l
emf2-l ld-1
emfl-l fca-1
emfl-l fd-1
42
60
59
27
>50%
>50%
>50%
emfl-l fve-1
emf2-lfe-l
emfl-l fy-1
emfl-l fha-1
emfl-l fpa-1
emfl-l co-1
emfl-l gi-1
31
65
31
48
54
55
29
WT (Col)
yes
yes
no
2.2 ±0.4
8.5 ±1.5
9.67 ±1.51
no
0
>SQ%
0
0
0
0
4.14±0.38
5.0 ±0.45
5.25±0.35
4.17±0.39
yes
yes
yes
yes
>50%
>50%
>50%
>50%
>50%
>50%
>50%
0
0
0
0
0
0
0
4.11 ±0.33
6.29±0.91
4.07 ±0.27
4.28±0.61
4.29+0.61
3.9 ±0.32
4.4 ±0.52
no
no
no
no
no
no
no
0
Number
of
flowers"
o
0
0
0
0
0
Fertility
Sepal
>35
>35
yes
yes
.54+0.52 yes
( ).2 ±1.10
O.55±O.3O ;-.43+0.53
0.83±0.39 :..0 ±0.63
0.6210.30 .40±0.54
0.82±0.40 .1.17±1.19
0
Flower structure
.22±0.67
!.07±0.62
;
.79±0.89
.29±0.47
.57+0.65
.0 ±0.00
.0 ±0.00
Petal Stamen
Carpel
yes
yes
no
yes
yes
yes
yes
yes
yes
yes
yes
no
sepal-petal
no
no
carpel-like
no
yes
yes
yes
yes
no
no
no
no
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
no
no
no
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
no
no
no
" The total number of plants scored.
* The average number of sessile leaves (small leaves without petiole) for plants at the time of floral bud emergence.
c
Branches subtended by cauline leaves.
d
The average number of flowers or flower-like lateral organs for plants.
inflorescence, and the number of flower produced.
Discussion
Although mutations in different late-flowering genes
cause similar phenotypes in delaying the onset of flowering,
our results clearly demonstrate that different late-flowering
genes interact with EMF genes differently. In general, each
late-flowering mutant allele causes a similar effect on both
emfl and emf2 mutant alleles. For example, fwa causes a
severe effect, whereas co causes no effect on both emf
mutants. This suggests that a particular late-flowering gene
interacts with two different EMF genes in a similar manner,
and supports the notion that two EMF genes have similar
functions.
Our results also indicate that late-flowering mutations
in the same group, previously characterized based on their
responses to environmental stimulations (Koornneef et al.
1991, Coupland 1995), cause a similar effect on both emf
mutations. For example, co and gi, both photoperiod and
vernalization insensitive, cause no effect on both emf
mutants. Mutations in FWA and FT, which are photoperiod sensitive and vernalization insensitive, cause the most
severe phenotype on both emf mutants. Mutations in lateflowering genes influenced by both changing daylength and
vernalization treatment, for example, FCA, FY, and LD,
have effects on emf mutations; however, this effect is in
relatively weak. This supports the notion that redundant
pathways do exist in response to environmental changes to
regulate flowering time in Arabidopsis. Genes involved in
the same or similar pathways, not surprisingly, cause
similar effects on emf mutants.
Double mutants in all combinations exhibit early emf
mutant phenotype, for example, they produced sessile
leaves or inflorescence directly from young seedlings. This
indicates that mutation in any late-flowering gene could not
restore the ability to form a normal rosette in emf plants,
and supports the notion that EMF genes are strictly required for rosette development. The result indicates that a
certain amount of EMF activity or the presence of both
EMF gene activity is necessary for the normal function of
all late-flowering genes tested during rosette development.
Low EMF activity or absence of one of the EMF functions
results in a bypassing of rosette development and premature formation of an inflorescence, no matter in the presence or absence of those late-flowering genes.
Unlike other late-flowering mutants, fwa strongly influence both the inflorescence and the flower structures in
double mutants in both strong and weak emf mutant
alleles. The complete elimination of any flower organs in
emfl-l fwa double mutants indicates that the mutant shoot
never becomes competent for flowering. The production of
Regulation of shoot development in Arabidopsis
392
leaf-like flowers in emf2 fwa double mutants indicate that
the formation of floral organs is altered. Since the increase
of leaf-shoot property in emffwa double mutants is similar
to the effect caused by leafy or apl mutations (Schultz and
Haughn 1993), these novel phenotypes indicate that FWA
is not only required for the early appearance of any flower
organs, but are also responsible for proper flower organ formation in both emf mutants. FWA seems to be strictly required for the proper expression of genes which specify
floral meristem identity. Without activation by FWA, the
activity of floral meristem identity genes will be reduced or
abolished, resulting either in the inability to initiate any
floral organs in fwa-1 emf I or in the formation of leaf-like
structures in fwa-1 emf2 double mutants. This is supported
by the results that fwa strongly enhanced both Ify and apl
mutant phenotype by producing only shoot structures without any flower formation in either fwa Ify/apl double
mutants (Madueno et al. 1996, Ruiz-Garcia 1997). This enhancement is much stronger than those caused by other
late-flowering mutants (Madueno et al. 1996, Ruiz-Garcia
1997). These results suggest that FWA represent a class of
late-flowering gene which regulates inflorescence development and activates floral meristem identity genes to
CO
GI
FT
FCA,LD, FD
FY, FVE,FE,
FPA, FHA,
EMF
Floral meristem
identity genes
Vegetative
FWA
Flowering
Fig. 5 Possible interactions between EMF and different lateflowering genes in regulating shoot development in wild-type
Arabidopsis. EMF genes function to activate (—•-) vegetative development and prohibit (—i) the expression of floral meristem
identity genes. The epistatic relationship between emf and two
late-flowering mutants co and gi suggests that CO and GI negatively regulate (—i) both EMF genes directly to promote flowering. FfVA may regulate flowering competence by inhibiting (—i)
vegetative development and activating (—-) floral meristem identity genes through distinct pathways from EMF genes. The other
nine late-flowering genes may function similarly to FWA but in a
relatively weak manner, or alternatively, they may be directly involved in the negative regulation (—i) of EMF genes to regulate
shoot maturation in Arabidopsis. In both cases, FT, LD, FCA,
and FD have stronger effect than do FY, FVE, FE, FPA, and
FHA.
regulate flowering time through distinct pathways from
both EMF and other late-flowering genes as represented in
Fig. 5.
The facts that the phenotype of both emf co and emf
gi double mutants is indistinguishable from emf single mutant confirm a previously published report that emf mutations are epistatic to co and gi (Yang et al. 1995). Since the
expression of CO has been reported to activate the transcription of LFY and API (Simon et al. 1996), CO and GI
may be involved in the regulation of shoot development
and activation of floral transition by negatively regulating
EMF directly (Fig. 5). Whether other late-flowering genes
interact with EMF genes directly or indirectly to regulate inflorescence and/or flower development remains uncertain.
However, since all other late-flowering mutants caused
almost no effect on strong emf mutant alleles, ie. emf 1-2
and emf2-l, we prefer that these late-flowering genes interact with EMF genes directly (Fig. 5). The additive phenotypes in inflorescence development observed in double
mutants between these late-flowering mutants and weak
emf mutant alleles, ie. emfl-1 and emf2-3, might result
from the inability of late-flowering mutant alleles to directly suppress the residual activity of a weak emf mutant
allele, thus causing the increase of the inflorescence growth
period. This assumption can be used to explain the reduction in frequency of flowering plants in emfl-1 double
mutants with several other late-flowering mutants such as
ld-1, fca-I and fd-1. The residual activity of the weak emfl1 allele may last longer in some fraction of double mutant
plants before senescence and result in the inability to form
any floral organs.
The various degree of the additive phenotypes observed in different double mutants may suggest that redundant pathways exist and reflect the different degree of interactions between these late-flowering mutants and two EMF
genes in regulating the shoot development. Since ft also influenced the flower structures by producing leaf or sepalcarpel intermediate structures in weak emf mutant allele
background and strongly enhanced both Ify and apl mutant phenotype in either ft Ify/apl double mutants (Madueno et al. 1996, Ruiz-Garcia 1997), therefore the pathway in which FT is involved may be more strictly needed
than LD and FCA, whereas LD and FCA are more strictly
needed than other late-flowering genes to negatively regulate EMF genes during shoot development (Fig. 5). Alternatively, the various effects of the additive phenotypes may
be due to the weak nature of some late-flowering mutant
alleles used in this study.
We thank the Arabidopsis Biological Resource Center for providing seed stocks used in this research. This work was supported
by a grant to C-H Y from the National Science Council, Taiwan,
Republic of China, grant number: NSC86-2313-B-OO5-O12.
Regulation of shoot development in Arabidopsis
References
Alvarez, J., Guli, C.L., Yu, X.H. and Smyth, D.R. (1992) terminal flower.
a gene affecting inflorescence development in Arabidopsis thaliana.
Plant J. 2: 103-116.
Amasino, R.M. (1996) Control of flowering time in plants. Current Opin.
Genet. Dev. 6: 480-487.
Araki, T. and Komeda, Y. (1993) Analysis of the role of the late-flowering
locus, GI, in the flowering of Arabidopsis thaliana. Plant J. 3: 231-239.
Bernier, G., Havelange, A., Houssa, C , Petitjean, A. and Lejeune, P.
(1993) Physiological signals that induce flowering. Plant Cell 5: 11471155.
Chen, L., Cheng, J - C , Castle, L. and Sung, Z.R. (1997). EMFgenes regulate Arabidopsis inflorescence development. Plant Cell 9: 2011-2024.
Clarke, J.H. and Dean, C. (1994) Mapping FRI, a locus controlling flowering time and vernalization response. Mol. Gen. Genet. 242: 81-89.
Clarke, J.H., Mithen, R., Brown, J.K.M. and Dean, C. (1995) QTL analysis of flowering time in Arabidopsis thaliana. Mol. Gen. Genet. 248:
278-286.
Coupland, G. (1995) Genetic and environmental control of flowering time
in Arabidopsis. Trends Genet. 11: 393-397.
Eimert, K., Wang, S-W., Lue, W-L. and Chen, J. (1995) Monogenic
recessive mutations causing both late floral initiation and excess starch accumulation in Arabidopsis. Plant Cell 7: 1703-1712.
Evans, L.T. (1969) The Induction of Flowering. Cornell University Press.
Goto, N., Kumagai, T. and Koornneef, M. (1991) Flowering responses to
light-breaks in photomorphogenic mutants of Arabidopsis thaliana, a
long-day plant. Physiol. Plant. 83: 209-215.
Hackett, W.P. (1985) Juvenility, maturation and rejuvenation in woody
plants. Horticult. Rev. 7: 109-155.
Halevy, A.H. (1985-1989) Handbook of flowering, Vols. I to VI. CRC
Press, Boca Raton.
Hicks, K.A., Millar, A.J., Carre, I.A., Somers, D.E., Straume, M., Ry
Meeks-Wagner, D. and Steve, A.K. (1996) Conditional circadian
dysfunction of the Arabidopsis early-flowering 3 mutant. Science 274:
790-792.
Koornneef, M., Hanhart, C.J. and van der Veen, J.H. (1991) A genetic
and physiological analysis of late flowering mutants in Arabidopsis
thaliana. Mol. Gen. Genet. 229: 57-66.
Lee, I., Bleecker, A. and Amasino, R.M. (1993) Analysis of naturally occurring late flowering in Arabidopsis thaliana. Mol. Gen. Genet. 237:
171-176.
Lee, I., Aukerman, M.J., Gore, S.L., Lohman, K.N., Michaels, S.D.,
Weaver, L.M., John, M.C., Feldmann, K.A. and Amasino, R.M. (1994)
Isolation of LUM1NIDEPENDENS: a gene involved in the control of
flowering time in Arabidopsis. Plant Cell 6: 75-83.
Madueno, F., Ruiz-Garcia, L., Salinas, J. and Martinez-Zapater, J.M.
(1996) Genetic interactions that promote the floral transition in Arabidopsis. Semin. Cell Dev. Biol. 7: 401-407.
Martinez-Zapater, J.M., Coupland, G., Dean, C. and Koornneef, M.
(1994) The transition to flowering in Arabidopsis. In Arabidopsis.
Edited by Somerville, C.R. and Meyerowitz, E.M. pp. 403-434. Cold
Spring Harbor Laboratory Press, New York.
393
Martinez-Zapater, J.M., Jarillo, J.A., Cruz-Alvarez, M., Roldan, M. and
Salinas, J. (1995) Arabidopsis late-flowering fve mutants are affected in
both vegetative and reproductive development. Plant J. 7: 543-551.
Martinez-Zapater, J.M. and Somerville, C.R. (1990) Effect of light quality
and vernalization on late-flowering mutants of Arabidopsis thaliana.
Plant Physiol. 92: 770-776.
McDaniel, C.N., Singer, S.R. and Smith, S.M.E. (1992) Development
states associated with the floral transition. Dev. Biol. 153: 59-69.
Medford, J.I., Behringer, F.J., Callos, J.D. and Feldmann, K.A. (1992)
Normal and abnormal development in the Arabidopsis vegetative shoot
apex. Plant Cell 4: 631-643.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-479.
Napp-Zinn, K. (1985) Arabidopsis thaliana. In Handbook of Flowering.
Edited by Halevy, A.H. pp. 492-503. CRC Press, Boca Raton.
Poethig, R.S. (1990) Phase change and the regulation of shoot morphogenesis in plants. Science 150: 923-930.
Putterill, J., Robson, F., Lee, K., Simon, R. and Coupland, G. (1995) The
CONSTANS gene of Arabidopsis promotes flowering and encodes a protein showing similarities to zincfingertranscription factors. Cell 80: 847857.
Ruiz-Garcia, L., Madueno, F., Wilkinson, M., Haughn, G., Salinas, J.
and Martinez-Zapater, J.M. (1997) Different role of flowering time genes
in the activation of floral initiation genes in Arabidopsis. Plant Cell 9:
1921-1934.
Sanda, S.L. and Amasino, R.M. (1996) Ecotype-specific expression of a
flowering mutant p h e n o t y p e in Arabidopsis
thaliana. Plant Physiol. I l l :
641-644.
Schultz, E.A. and Haughn, G. W. (1993) Genetic analysis of the floral initiation process (FLIP) in Arabidopsis. Development 119: 745-765.
Shannon, S. and Meeks-Wanger, D.R. (1991) A mutation in the Arabidopsis TFL1 gene affects inflorescence meristem development. Plant Cell
3: 877-892.
Shannon, S. and Meeks-Wanger, D.R. (1993) Genetic interactions that regulate inflorescence development in Arabidopsis. Plant Cell 5: 639-655.
Simon, R., Igeno, M.I. and Coupland, G. (1996) Activation of floral
meristem identity genes in Arabidopsis. Nature 384: 59-62.
Sung, Z.R., Belachew, A., Bai, S. and Bertrand-Garcia, R. (1992) EMF,
an Arabidopsis gene required for vegetative shoot development. Science
258: 1645-1647.
Vince-Prue, D. (1983) Photomorphogenesis and flowering. In Photomorphogenesis. Edited by Shropshire, W., Jr. and Mohr, H. pp. 457-490.
Springer-Verlag, Berlin.
Wilson, R.N., Heckman, J.W. and Somerville, C.R. (1992) Gibberellin is
required for flowering in Arabidopsis thaliana under short days. Plant
Physiol. 100: 403-408.
Yang, C-H., Cheng, L-J. and Sung, Z.R. (1995) Genetic regulation of
shoot development in Arabidopsis: the role of EMF genes. Dev. Biol.
169: 421-435.
Zagotta, M.T., Shannon, S., Jacobs, C. and Meeks-Wagner, R. (1992) Early- flowering mutants of Arabidopsis thaliana. Aust. J. Plant Physiol.
19: 411-418.
(Received October 18, 1997; Accepted January 27, 1998)