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Plant CellPhysiol. 41(1): 94-103 (2000)
JSPP © 2000
Isolation and Characterization of an Arabidopsis Mutant, fireworks (fiw),
Which Exhibits Premature Cessation of Inflorescence Growth and Early Leaf
Senescence
Masanobu Nakamura, Nobuyoshi Mochizuki and Akira Nagatani
Laboratory of Plant Physiology, Department of Botany, Kyoto University, Kitashirakawa-Oiwake-Cho, Sakyo-Ku, Kyoto, 606-8502
Japan
trast, the reproductive shoot or inflorescence is characterized by long stems, from which lateral cauline leaves,
inflorescences, and solitary flowers emerge (Schultz and
Haughn 1991). In the final stage of the development, inflorescence sets seeds and stops growing. Although the
Arabidopsis inflorescence is structurally indeterminate and
could develop an unlimited number of flowers, the actual
shoot apical meristem stops proliferation after developing
a predictable number of flowers (Shannon and MeeksWagner 1991, Bleecker and Patterson 1997).
Senescence means whole-plant senescence in the life
history of annual plants like Arabidopsis, and is coupled
with monocarpic reproduction (Nooden and Leopold 1988,
Bleecker and Patterson 1997, Nooden et al. 1997). The
process includes cessation of elongation growth and inflorescence development, leaf yellowing and shedding
(Nooden 1980, Thimann 1980, Halliday et al. 1996). To
maximize its fitness, which means setting the maximum
number of healthy seeds, vegetative tissues break down
themselves partially, enlower their running costs, and
translocate the resultant nutrients to seeds and reproductive tissues (Bleecker and Patterson 1997, Nooden et al.
1997). In this way, the vegetative tissue acts as a source in
a later stage of development.
In the process of whole-plant senescence, different
parts of plants should senesce coordinately. How is this
highly organized process regulated then? This question has
long been argued and models have been proposed through
many physiological analyses (Gan and Amasino 1997,
Hensel et al. 1993, Kelly and Davies 1988, King et al. 1995,
Nooden and Leopold 1988). In soybean and other monocarpic species, it has been shown that reproductive structures such as flowers and siliques could control the monocarpic senescence (Hamilton and Davies 1988). However,
analysis of late-flowering and male-sterile mutants in
Arabidopsis suggests that somatic tissue longevity is not
under control of these structures in Arabidopsis (Hensel et
al. 1993, Bleecker and Patterson 1997). Although some
substance might act as a "death hormone", such a substance has never been identified.
Genetic approach is a powerful tool to unravel the
molecular mechanisms of various biological processes.
With respect to inflorescence development, various mutants with short inflorescence have been isolated (Koorn-
To examine the mechanism underlying the reproductive development in monocarpic plants, we screened for
mutants that exhibit premature cessation of inflorescence
growth in Arabidopsis. We identified a novel mutant line
that exhibited earlier cessation of flower formation and
inflorescence stem elongation. This mutant also exhibited
accelerated rosette leaf senescence after the cessation of the
inflorescence growth. We designated the mutant fireworks
(fiw) because flowers and siliques were clustered at the top
of the fiw inflorescence. The fiw mutation was a single,
recessive mutation and mapped on the lower part of chromosome 4. The fiw phenotype was not observable during
vegetative growth, but the inflorescence growth was arrested more than 7 d earlier than the wild type (WT). Microscopic observation revealed that the fiw apical meristem
was structurally preserved. The premature arrest of growth
was observed not only in the primary inflorescence but also
in the lateral inflorescence, which is consistent with the
global proliferative arrest observed later in WT. Regardless
of such dramatic phenotypic features, the fiw plants bore
normal flowers and set fully matured siliques.
Key words: Arabidopsis thaliana — Dwarf — Meristematic
activity — Mutant — Stem elongation — Whole-plant
senescence.
In the life cycle of rosette plants such as Arabidopsis,
the developmental pattern of shoots changes dramatically
before and after the onset of the reproductive growth.
During the vegetative phase, a shoot is in a compressed
form (rosette) and its internodes do not elongate. In conAbbreviations: ACL, ACAULIS loci in Arabidopsis; CAPS,
cleaved, amplified, polymorphic sequences; Col, Columbia ecotype of Arabidopsis; ER, ERECTA locus in Arabidopsis; fiw,
fireworks mutant of Arabidopsis; GPA, global proliferative arrest; Ler, Landsberg erecta ecotype of Arabidopsis; SSLP, simple
sequence length polymorphism; T-DNA, transferred-DNA; TFL,
TERMINAL FLOWER locus in Arabidopsis; WT, wild type.
Correspondence to: Akira Nagatani, Laboratory of Plant
Physiology, Department of Botany, Kyoto University, Kitashirakawa-Oiwake-Cho, Sakyo-Ku, Kyoto, 606-8502 Japan.
Tel, +81-75-753-4123; Fax, +81-75-753-4126; e-mail, nagatani®
physiol.bot.kyoto-u.ac.jp
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Inflorescence growth and senescence mutant
neef et al. 1985, Shannon and Meeks-Wagner 1991, Bowman 1993, Kieber et al. 1993, Tsukaya et al. 1993, Szekeres
et al. 1996). Most of them are categorized to be hormonal
mutants (Bowman 1993, Li et al. 1996, Szekeres et al.
1996). These studies clearly indicate the importance of
plant hormones for normal development of inflorescence.
Nevertheless, these mutants provide us little information
about the mechanism of whole-plant senescence. No sole
plant hormone appears to be able to trigger the whole
process, although some hormones clearly affect the process
(Zacarias and Reid 1990, Grbic and Bleecker 1995,
Bleecker and Patterson 1997, Gan and Amasino 1995).
Some mutants such as acaulis (act) (Tsukaya et al.
1993, 1995, Hanzawa et al. 1997) and terminal flowerl
(tfll) (Shannon and Meeks-Wagner 1991) show earlier cessation of inflorescence growth. The flower stalks of acl
mutants (acll, acl2 and acl5), which are almost absent or
are much reduced in length, exhibit premature arrest of
reproductive development, which is followed by consequent reduction in the number of flowers. However, inflorescence meristems are structurally indeterminate in acl
mutants (Tsukaya et al. 1993, 1995, Hanzawa et al. 1997).
On the other hand, the inflorescence development of tfll-1
mutant is limited by production of a terminal floral meristem. In addition, the erecta mutation, which produces a
compact inflorescence, is present in a Landsberg erecta
(Ler) genetic background (Bowman 1993, Torii et al.
1996). The senescence phenotype of these mutants has not
been examined intensively, but leaf senescence does not
appear to be affected.
To elucidate the mechanism underlying the inflorescence development, especially in the context of the wholeplant senescence, we screened for mutants that exhibit
premature cessation of inflorescence growth. We isolated
one recessive mutant line designated fireworks (fiw). In the
fiw mutant, the primary as well as lateral inflorescence
meristem stopped proliferation earlier and elongation of
inflorescence internodes was severely inhibited. Interestingly, accelerated leaf senescence was observed as well in
this mutant. Despite these dramatic phenotypic features,
the fiw mutant did not show any characteristic phenotype
during the vegetative phase. Further analysis of this mutant
should provide us new insights into a highly complex
senescing syndrome in monocarpic plants.
95
(Boehringer, Germany). The culture medium was solidified with
0.6% (w/v) of Phytagar (Gibco BRL). Seeds were soaked in the
basal culture medium and cultured for 15 d. Seedlings were then
transferred to rockwool moistened with hydroponic medium
(Tsukaya et al. 1991). All the plants were grown in environmentally controlled growth chambers under continuous white light at
22°C except in the temperature shift-up experiments.
Mutagenesis of Arabidopsis plant—Agrobacterium-mediated
transferred-DNA (T-DNA) mutagenesis was performed using
28-day-old her plants (TO plant) by the in planta transformation method (Bechtold et al. 1993). Transformation vector
pPCVICEn4HPT was a kind gift from Dr. R. Walden (Hayashi et
al. 1992). About 800,000 seeds (Tl seeds) harvested from TO
plants were plated on the basal culture media containing 22.5 fig
m P 1 of hygromycin B. About 1,000 hygromycin-resistant Tl
plants were selected and grown for further analysis. For establishment of mutagenized T2 lines, selected Tl plants were selfed
and seeds were collected separately from each plant.
Screening for short inflorescence mutation—T2 seeds of individual lines were grown to screen short inflorescence and early
senescence mutants. One mutant line which showed a severe
defect in inflorescence stem elongation was identified and designated fireworks (fiw). To characterize the fiw mutant, we backcrossed the T2 generation of fiw mutant twice to the wild type
(WT).
Morphological observations—Bolting plants grown as described above were used for size measurements. All measurements
were made on several different plants. To follow the time course
of flower formation, shoot apices were photographed every day
and developing flowers and flower buds emerged directly from the
primary inflorescence stems were numbered in the order of appearance. Inflorescence stems used for histological studies were
obtained from plants grown under the same conditions. For microscopic analysis, stems and apical buds were fixed and cleared as
described by Aida et al. (Aida et al. 1997).
Chromosome mapping—A single plant showing the fiw
phenotype (her background) was pollinated with Col pollen. A
resultant Fl hybrid was selfed to generate a segregation F2
population of 57 mutants and 175 WT individuals. Small-scale
preparation of plant genomic DNA was done by the method of
Edwards et al. (Edwards et al. 1991). Linkage to known molecular
markers was determined by simple sequence length polymorphism
(SSLP) (Bell and Ecker 1994) and cleaved, amplified, polymorphic
sequences (CAPS) (Konieczny and Ausubel 1993) using isolated
individual genomic DNA and oligo nucleotide primers synthesized
by Biologica (Nagoya, Japan).
Chlorophyll analysis—Chlorophyll was extracted from the
fresh 6th rosette leaves with N,N,-dimethyl formamide. The extracts were subjected to spectrophotometric measurements at 664
and 647 nm. Chlorophyll contents were calculated using the equation of Moran (Moran 1982).
Results
Materials and Methods
Plant materials—The following plants were used: Arabidopsis thaliana (L.) Heynh. ecotype Landsberg erecta (her) and
Columbia (Col). Seeds were surface sterilized in a solution of
NaCIO ( C l > 1 . 7 % , 0.02% (v/v) Triton X-100) for 1 min and
rinsed extensively with distilled water. Basal culture medium was
composed of Murashige-Skoog inorganic salts (Murashige and
Skoog 1962) supplemented with 2% (w/v) sucrose, B5 vitamins
(Gamborg et al. 1968), and with or without hygromycin B
Overall morphology of the fiw mutant—To better
define the mechanisms involved in the control of the last
stage of monocarpic development, we isolated an Arabidopsis mutant in which both cessation of inflorescence
growth and leaf senescence occurred earlier. The mutant
was designated as fireworks (fiw) after its characteristic
inflorescence appearance. Fig. 1A and B show the outlook
of 30-day-old plants of ihe fiw mutant and WT (her). The
96
Inflorescence growth and senescence mutant
fiw mutant had a much shorter inflorescence than WT,
suggesting that the inflorescence growth was arrested much
earlier in the fiw mutant. The growth of lateral inflores-
cence was suppressed as well. In addition, rosette leaves
and other parts of the fiw mutant senesced much earlier in
the fiw mutant than in WT (Fig. 2). Parts of rosette leaves
Fig. 1 Phenotype of fiw mutant and WT. Side view of 30-day-old plants of fiw mutant (A) and WT (B). Top view of 33-day-old fiw
mutant (C) and 33- (D) or 43- (E) day-old WT plant. (F): Magnified view of the topmost parts of the fiw mutant (left) and WT (right)
inflorescence. Lateral shoots and flowers were removed from the stems of 33-day-old ./zw mutant or WT. Numbers indicate the order of
flowers formed on the primary inflorescence stem. Photographs of fiw mutant in er (G) and ER (H) background. F2 fiw/fiw segregants
from Fl hybrid between the fiw mutant (er/er, fiw/fiw) and Columbia WT (ER/ER, FIW/FIW) with or without er mutation were
photographed. The ER plants were recognized by shapes of siliques and leaves and peduncle length. Bar=l cm (A, B, G and H), bar=l
mm (C, D, E and F).
Inflorescence growth and senescence mutant
started to turn yellow or brown already around day 41 in
the mutant (Fig. 2C), whereas the leaves appeared somewhat paler but still green even on day 47 in WT (Fig. 2F).
Top view of the apical region of the fiw mutant
(Fig. 1C) indicated that it had less flower buds than WT at
the same age (Fig. ID). In theyhv apex, only small flower
buds were observed. These flower buds did not grow any
further. Hence, the fiw apex resembled an aged inflorescence apex of WT (Fig. IE). In addition, severe inhibition
of internode elongation was observed in the topmost region
of t\\Q fiw apex (Fig. 1C, F). As a result, mature siliques
were clustered at the top of the fiw inflorescence. In contrast, development of flower buds and internode elongation
appear to be coordinated in WT. A full series of developing
flowers and fruits were placed at adequate intervals on WT
stem even after the cessation of inflorescence growth
(Fig. ID, E). Irrespective of these dramatic features at the
reproductive stage, the fiw mutant was indistinguishable
from WT at the vegetative stage (see below).
Genetic analysis of the fiw mutation—The fiw mutants
were backcrossed to WT Ler or Col. The Fl resembled
WT in appearance. The F2 population segregated in a 3 : 1
(WT : mutant) ratio, suggesting that the fiw mutation was
caused by a single recessive Mendelian allele (Table 1).
Although the fiw mutant was isolated from T-DNA mutagenized Arabidopsis lines, it was not linked to a T-DNA
insert (data not shown). Thus, the characterization of the
fiw mutant in this study was done using backcrossed, TDNA non-bearing plants.
To determine the map position of the FIW locus, genomic DNA was isolated from mutant segregants among
Fig. 2 Leaf senescence of fiw mutant and WT. Rosette leaves of
fiw mutant (A, C, E), and WT (B, D, F) were photographed on
day 35 (A, B), 41 (C, D) and 47 (E, F).
97
Table 1 Phenotypic ratios in populations of fiw/fiw x
FIW/FIW and fiw/FIW * fiw/FIW crosses
Phenotype
Crosses
fiw/fiw*
FIW/FIW
fiw/FIW* fiw/FIW
WT
fiw
Ratio
20
0
1:0
134
42
3.19: 1
the F2 population of a fiw x Col cross and analyzed
for linkage to SSLP (Bell and Ecker 1994) and CAPS
(Konieczny and Ausubel 1993) markers. No linkage between the fiw phenotype and the following SSLP markers
was observed: nga63 and nga280 on chromosome 1; ngal68
on chromosome 2; ngal62 and nga6 on chromosome 3;
nga225 and nga76 on chromosome 5. The fiw phenotype,
Fig. 6 Photographs of apical regions of the fiw and WT
inflorescence. The apical regions of the fiw mutant (A, C, E) and
WT (B, D, F) were photographed from above on day 20 (A, B), 26
(C, D) and 31 (E, F). Flowers emerged directly from the primary
inflorescence were numbered in the order of appearance. Flowers
and flower buds with a diameter of over 300 /um were numbered.
98
Inflorescence growth and senescence mutant
however, was found to be slightly linked to nga8 on chromosome 4 (data not shown). Of the CAPS markers examined, fiw was mapped between two CAPS markers B9
and PG11 on chromosome 4 (data not shown).
There is no other related mutation around this map
region except acaulisl (acll) (Tsukaya et al. 1993). Then,
the allelism test between acll-2 and fiw was performed. The
result indicated that ACL1 and FIW are independent loci
(data not shown). So, we concluded that fiw is a novel locus.
The ecotype her, which is the background of the fiw
mutant, includes an endogenous dwarf mutation named
erecta (er) (Torii et al. 1996). Thus, the original fiw line we
isolated should be a fiw er double mutant. To reveal the
genetic interaction between the FIW and ER loci, we
10
20
30
40
Total number of flowers
50
crossed the fiw mutant with Col to find that the fiw
phenotype was independent of the ER locus. Nearly onefourth of the F2 progeny from Fl hybrid of fiw x WT Col
exhibited the fiw phenotype (data not shown). Moreover,
these F2 fiw phenotype populations showed segregation in
the er phenotype (Bowman 1993) (Fig. 1G, H).
Developmental parameters of the fiw mutant—Fig. 3A
shows the relationship between the number of flowers
and the height of the primary inflorescence stem in the
fiw mutant and WT. The fiw mutant set fewer flowers
(19.0±4.1) on its shorter stems (65.6±26.7 mm) than WT
(flowers, 33.6±3.4; inflorescence stems, 206±19.0mm).
Although the number of flowers and the length of inflorescence stem varied significantly among the fiw individuals, they could be clearly distinguished from WT
(Fig. 3A). Similar analysis on the upper part of the stem
(from the topmost cauline leaf to the shoot apex) indicated
that the reduction in elongation was much severer in the
upper part (Fig. 3B). The length of the upper part of the fiw
mutant was nearly 10% of that in WT (fiw, 12.5 ±4.8 mm;
WT, 118±18.8mm). Since the variation among the fiw
individuals was large, the plants with average sizes were
chosen for further analysis.
The fiw mutant was compared with WT with respect to
several developmental parameters (Table 2). The number
and size of rosette leaves were unaffected in the fiw
mutants. The timing of the onset of inflorescence stem
elongation, which reflects the timing of transition from
vegetative to reproductive stage, was indistinguishable between the fiw mutant and WT. The number of cauline
leaves was unaffected as well in the fiw mutant. Thus, the
phenotype of the fiw mutant appears to be restricted to
the reproductive stage. However, not all the aspects*of
reproductive growth were affected in the fiw mutant.
Growth of siliques was not disturbed in the fiw mutants.
The size of flowers and peduncles were normal as well (data
Table 2 Sizes and number of different organs in WT and
fiw plants
10
20
30
40
50
fiw
WT
18.4±0.5
18.8 + 0.8
8.6±0.5
8.9±0.9
Size of 6th rosette leaves (mm)
length
24.5±3.8
width
11.1 ±1.9
26.1±3.9
12.1 + 1.5
Total number of flowers
Fig. 3 Relationship between the number of flowers and the inflorescence length in the fiw mutant or WT individuals. Total
number of flowers vs. lengths of primary inflorescence (A) and
total number of flowers vs. lengths of upper part of primary inflorescence stem (B) were plotted. Upper part was defined here as
the part of stem above the topmost cauline leaf. Solid circles, fiw
mutant; solid triangles, WT. Each point represents an individual
plant. The number of flowers was counted only for those directly
emerged from primary inflorescence stems. Flowers and flower
buds with a diameter of over 300 /urn corresponding to stage 9 or
later as defined by Smyth et al. (Smyth et al. 1990) were counted.
Numbers of flowers and lengths of stems were determined at least
several days after the cessation of inflorescence growth.
Bolting time (day)
a
Number of rosette leaves
a
Number of cauline leavesa
3.3±0.5
3.3±0.8
Length of siliques * (mm)
9.5±0.7
9.4±1.0
a
Measured 30 d after germination.
* Measured 45 d after germination. Each value represents the
average±standard error of at least 10 plants.
Inflorescence growth and senescence mutant
not shown). The fiw mutants set fully fertile flowers although the number of flowers was reduced.
Stem elongation—To further characterize the fiw
phenotype, we examined the time course of the inflorescence stem elongation (Fig. 4A). The primary inflorescence
initiated elongation around day 19 after germination both
in the fiw and WT plants. However, the rate of growth
started to decrease around day 25 and the growth completely stopped around day 29 in the fiw mutant. In contrast, WT stems continued to grow until about day 36. As
shown in Fig.4B, the upper parts of the stem (from the
topmost cauline leaf to the shoot apex) of both the fiw and
WT initiated elongation around day 24. However, the
elongation rate was greatly reduced in the fiw mutant from
the beginning. The elongation ceased about 7 d earlier in
the fiw mutant than in WT. Thus, the initiation of inflorescence growth was not affected in the fiw mutant but
the cessation of growth occurred earlier in the fiw mutant.
Next we observed epidermal cell layers of the fiw and
WT inflorescence stems. As shown in Fig. 5A and B, the
longitudinal cell length in the fiw mutant was greatly
reduced compared with WT. In contrast, cell width was not
99
affected in the fiw mutant. These results are consistent with
the gross morphology of the fiw inflorescence, namely,
longitudinal elongation of the stem was severely inhibited
whereas radial expansion appeared relatively normal
(Fig. IF).
Flower bud formation—The number of flowers was
reduced in the fiw mutant (Table 2). Representative photographs of the shoot apices at different time points are
shown in Fig. 6A-F. Shoot apices of 20-day-old fiw plants
were indistinguishable from those of WT (Fig. 6A, B), and
the number of flowers (flower buds) was nearly the same.
On day 26, the number of flowers and the area of shoot
apices covered with flower buds were slightly smaller in the
fiw mutant compared with WT (Fig. 6C, D). Coincidentally, the elongation rate of fiw stems slowed down around
day 25 (Fig. 4). On day 31, the difference between the fiw
mutant and WT was obvious (Fig. 6E, F). Mature flowers
with normal size pedicels were clustered at the top of the
fiw inflorescence. The fiw apex totally lacked medium-sized
flower buds, although small buds were present (Fig. 6E). In
contrast, WT apex still bore a full series of developing
flowers on day 31, although the area of shoot apex became
slightly smaller than that on day 26 (Fig. 6D, F). As shown
in Fig. 4, the fiw primary inflorescence had already ceased
elongation at this time point, while WT inflorescence was
still growing.
38
Fig. 4 Elongation growth curves of the primary inflorescence of
fiw mutant and WT. Total height (A) and the length above the
topmost cauline leaf (B) of the primary inflorescence were measured daily. Solid circles, fiw mutant; solid triangles, WT. Vertical
bars indicate standard errors. All measurements are the means of
at least 4 different plants.
Fig. 5 Microscopic observations of epidermis of the fiw mutant
and WT stems. Differential interference contrast microscopic images of epidermal cell files of the primary inflorescence stem of the
fiw mutant (A) and WT (B). Fifth internodes above the topmost
cauline leaf of 30-day-old plants were observed. Bar=50/wm.
100
Inflorescence growth and senescence mutant
Fig. 7 Time course analysis of flower formation on the primary
inflorescence. The number of flowers were plotted against age.
Plots are the results of a single plant which is a representative of
either a.fiwmutant or WT in its elongation growth. Solid circles
indicate data from fiw mutant and solid triangles indicate data
from WT (materials of these plots were not the same as those used
in Fig. 6).
The number of the flowers that emerged directly from
the primary inflorescence stems was counted during the
course of stem elongation. Representative data are shown
in Fig. 7. In the early stage of inflorescence development,
the fiw mutant formed flower buds at almost a normal rate.
However, the mutant plants stopped to form flowers a few
days before cessation of stem elongation (Fig. 4, 7). In
contrast, WT plants stopped flower formation much later
(Fig. 7). Thus, we concluded that the growth arrest of the
primary inflorescence meristem occurs earlier in the fiw
mutant.
To determine whether the inflorescence meristem of
the fiw mutant was structurally altered, we observed the
apical region under a microscope (Fig. 8A, B). Apices of
the fiw and WT plants were fixed at day 30 and cleared for
the observation. At this time point, the fiw mutant had
already ceased flower formation (Fig. 6E, 7), whereas many
flower buds were present on the shoot apex and flower
formation was still in progress in WT (Fig.6F, 7). Regardless of the big difference in the gross appearance
of shoot apices of the fiw mutant and WT (Fig. 6), the
meristematic region of the fiw mutant appeared to be quite
normal (Fig. 8). The inflorescence meristem was similar
in size to that of WT. The number and arrangement of
flower primordia (<50//m) were indistinguishable from
those of WT. These observations suggest that the fiw mutation does not alter the structure of inflorescence meristem.
Lateral inflorescence growth—In WT, elongation of
lateral inflorescences started around day 25, which was
about 6 d after the onset of the primary inflorescence
elongation. The elongation growth of lateral inflorescences
stopped within a few days after the cessation of primary
inflorescence growth (representative data are shown in
Fig. 9A). This phenomenon is referred to as global proliferative arrest (GPA) of inflorescence meristems (Hensel et
al. 1994). Thus, we examined whether the same mechanism
is working in the fiw mutant. Elongation of lateral inflorescences also started around day 25 in the fiw mutant.
The elongation then stopped within a few days. As a result,
most of the lateral inflorescences reached only 0-6 mm long
(representative data are shown in Fig.9B). Thus, the ces-
^250
-•—primary
-•-lateral 1
- A - lateral 2-1
-M-lateral 2-2
—t—lateral 2-3
0 *•<
18 20 22 24 26 28 30 32 34 36 38 40
Days
B
250
- primary
-lateral 1
-lateral 2-1
-lateral 2-2
-lateral 2-3
18 20 22 24 26 28 30 32 34 36 38 40
Days
Fig. 8 Magnified view of apical regions of fiw and WT inflorescence. Differential interference contrast microscopic images of
apical regions of fiw mutant (A) and WT (B), at 30 d old. Bar = 50
Fig. 9 Time course analysis of the elongation of inflorescence
stems. Length of primary and lateral inflorescence stems was
measured daily. Data from representative individuals were plotted
(A; WT, B;fiw). Lateral 1, a lateral inflorescence subtended by a
rosette leaf. Lateral 2-1 to 2-3, lateral inflorescence emerged from
the first to third inflorescence nodes.
Inflorescence growth and senescence mutant
15 20 25 30 35 40 45 50 55 60 65
Days
Fig. 10 Time course analysis of chlorophyll content. Chlorophyll was extracted from the 6th rosette leaves. Solid circles
indicate data from fiw mutant and solid triangles indicate data
from WT. Vertical bars indicate standard errors. All measurements were made on at least 3 individual plants.
sation of growth of the primary inflorescence and lateral
inflorescences occurred almost simultaneously in the fiw
mutant, which is consistent with the GPA model.
Leaf senescence—As shown in Fig. 2, leaf senescence
occurred earlier in the fiw mutant. To quantify this phenomenon, we measured the chlorophyll content of rosette
leaves during the course of senescence (Fig. 10). The results
indicated that a rapid decrease in the chlorophyll level in
the fiw started as early as around day 30. In contrast, WT
leaves still contained a certain amount of chlorophyll on
day 60, although a moderate decrease in the chlorophyll
content was observed over the period of the measurement
(Fig. 10).
Discussion
In this report, we have described the initial characterization of an Arabidopsis short inflorescence and early
senescence mutant, fiw. The fiw phenotype was due to a
nuclear, recessive mutation (Table 1). Unlike other dwarf
mutants, growth defects in the fiw mutant were restricted
to a later stage of development. The timing of transition
from the vegetative to reproductive stage was not altered.
Nevertheless, it exhibited clear pleiotropic phenotype in the
reproductive stage. Elongation of the inflorescence stems
including lateral ones was arrested much earlier in the fiw
mutant. Flower formation stopped earlier as well. In addition, its rosette leaves senesced much earlier than those in
WT.
The fiw mutant and other inflorescence stem mutants
—Genetic analysis of the fiw mutant suggested that it
defines a novel locus. The fiw mutation was mapped between two CAPS markers B9 and PG11 on chromosome 4
101
(data not shown). No related mutation is known around
this map region except acll, (Tsukaya et al. 1993). An allelism test between acll-2 and fiw confirmed that ACL1 and
FIW are independent loci.
The acl mutants {acll, 2 and 5) resemble the fiw
mutant in some ways (Tsukaya et al. 1993, 1995, Hanzawa
et al. 1997). These mutants exhibit earlier growth arrest of
the inflorescence. However, there are clear differences between the fiw and acl mutants. The elongation defect of fiw
was limited to the stem (Table 2). In contrast, acl mutations affect elongation growth in both vegetative and
reproductive organs (Tsukaya et al. 1993, 1995, Hanzawa
et al. 1997). In addition, the acll and acl2 mutations are
complemented by a temperature shift-up from 16°C to
28°C, whereas the fiw phenotype was not temperature dependent (data not shown).
Arabidopsis ecotype Landsberg includes a kind of
dwarf mutation called erecta (er), which alters the morphology of the inflorescence stem (Bowman 1993, Torii et
al. 1996). We examined the relationship between the FIW
and ER loci genetically. The cross experiment between fiw
(Ler background) and Col revealed that there is no strong
interaction between these two loci (Fig. 1G, H).
Plant hormones and the fiw mutant—Plant hormones
affect various aspects of plant development. Mutants
aberrant in the hormonal states exhibit pleiotropic phenotype. Some hormonal mutants exhibit the dwarf phenotype (Koornneef and van den Veen 1980, Bowman 1993).
However, growth defects are observed in multiple organs
throughout the life cycle in such mutants. In addition, a
number of dwarf mutants, including hormone-related
ones, show reduced apical dominance (Estelle and Somerville 1987, Chory et al. 1991, Clouse et al. 1996). In contrast, growth defects in the fiw mutant are restricted to
a certain stage of development. The growth of lateral
inflorescence stems is suppressed rather than enhanced in
the fiw mutant (Fig. IB, 9). Thus, it is not likely that the fiw
mutant is a simple hormonal mutant.
Exogeneously applied gibberellic acid induces internode elongation (Jacobsen and Olszewski 1993). To test
whether gibberellin can restore the fiw phenotypic features,
the fiw plants were grown in the presence of exogenous
gibberellin. Although gibberellin induced stem elongation
in the fiw mutant, the extent of the response was comparable to that observed in WT (data not shown). Neither
severe deficiency in the internode elongation at the topmost
part of the stem nor the early leaf senescence phenotype
was restored by gibberellin. Hence, the gibberellin defect
does not appear to be the primary cause of the fiw phenotype.
Some plant hormones control leaf senescence. Treatment of Arabidopsis plants with ethylene or abscisic acid
promotes the premature onset of the senescence syndrome
(Zacarias and Reid 1990, Grbic and Bleecker 1995, Ble-
102
Inflorescence growth and senescence mutant
ecker and Patterson 1997). On the contrary, elevated
cytokinin delays leaf senescence (Gan and Amasino 1995).
Therefore it is possible that some hormonal derangement
has occurred in the fiw mutants. However, it is difficult to
explain the fiw phenotype simply by the deficiency in one of
these hormones. Mutants deficient in these hormones exhibit pleiotropic phenotype throughout the life cycle (Kieber et al. 1993, Chaudhury et al. 1993, Leon-Kloosterziel et
al. 1996), whereas the fiw phenotype is strictly restricted to
the later stage of development.
Apical morphology and meristem identity of the fiw
mutant—One of the inflorescence mutants, tfll-1 (Shannon
and Meeks-Wagner 1991), shares some phenotypic features
in common with the fiw mutant. Both mutants have shorter
inflorescence stems and set fewer solitary flowers. However, the mechanism causing such a phenotype appears to
be different in these mutants. In tfll-1, inflorescence, which
is normally indeterminate in Arabidopsis, forms the terminal floral meristem. In contrast, the central region of the
apical meristem of the fiw mutant remained domed and
undeterminated even after the cessation of inflorescence
development (Fig. 8). In addition, the tfll-1 mutant shows
an early flowering phenotype which was not observed in the
fiw mutant (Shannon and Meeks-Wagner 1991). Thus, the
FIW protein appears to be involved neither in the establishment of inflorescence meristem nor the maintenance of
its indeterminate state.
The term GPA was proposed by Hensel et al. (Hensel
et al. 1994) referring to the natural growth cessation observed in Arabidopsis ecotype Ler after setting a predictable number of flowers on the primary inflorescence meristem (Shannon and Meeks-Wagner 1991). Two major
features of GPA are (1) the structure of the shoot apex is
preserved even after GPA. The arrested inflorescences have
a potential to re-initiate proliferation (Bleecker and Patterson 1997), and (2) the growth of the primary and lateral
shoot apical meristems is arrested simultaneously within a
few days. As discussed above, the shoot apex of fiw mutants remained normal in size and phyllotaxis even after
cessation of growth (Fig. 8). Lateral inflorescence stems
ceased to grow within a few days after the cessation of
primary inflorescence stem elongation (Fig. 9). Thus, it
appears as if GPA occurred earlier in the fiw mutant.
However, some phenotypic features of the fiw mutant
can not be explained simply by the accelerated GPA. Floral
development and internode elongation coordinately cease
during the course of GPA in WT. As a result, the shoot
apex bears flowers and flower buds of various sizes at
normal intervals. In contrast, flower buds that exceeded a
certain size continued to grow even after the cessation of
internode elongation in the fiw mutant, which resulted in
the appearance characterized by many siliques clustered at
the top of the stem. In addition, WT plants do not exhibit
GPA when developing siliques are continuously removed
(Hensel et al. 1994). However, this phenomenon was not
observed in the fiw mutant (data not shown).
Relationships between the fiw pleiotropic phenotypic
features—The phenotype of the fiw mutant included earlier cessation of internode elongation, earlier cessation of
flower formation at the shoot apex, and earlier leaf senescence. These three major phenotypic features became visible more or less simultaneously. The reduction in the rate
of stem elongation was recognized around day 25 after
sowing (Fig. 4). The number of solitary flowers ceased to
increase around day 26 (Fig. 7). The decrease of chlorophyll content in rosette leaves started around day 30
(Fig. 10). Among them, leaf senescence appears to start a
little later than the others. However, the onset of the
reduction in protein and carbohydrate contents precedes
the decrease in leaf chlorophyll by several days (Hensel et
al. 1993). Thus, it is likely that some process of leaf
senescence already started several days before day 30 in the
fiw mutant. Thus, it is difficult to speculate about the
causal relationships between these phenotypes. One intriguing possibility is that the fiw mutation affects a key
event which triggers these three processes.
Although the fiw mutation caused earlier cessation of
internode elongation and flower formation, the growth of
individual flowers appeared to be less affected. When GPA
occurs, flowers and flower buds appear to cease to grow
coordinately in WT. In contrast, flower buds that exceeded
a certain size continued to grow after the cessation of the
apical growth in the fiw mutant. Thus, the growth of the
shoot apex and flower buds appears to be uncoupled to
some extent in the fiw mutant.
The analysis of late-flowering and male-sterile mutants
in Arabidopsis suggested that somatic tissue longevity is
not governed by reproductive development (Hensel et al.
1993). However, both the leaf senescence and proliferative
arrest of meristem started earlier in the fiw mutant. Hence,
there might be a common mechanism which regulates both
processes. Alternatively, an acute arrest of growth at the
apex may have caused abnormal leaf senescence through an
unusual pathway or vice versa. In any case, grafting experiments in future should help determine which part of
the plant generates the signal to senesce.
We thank Dr. Y. Komeda (Hokkaido Univ.) for kindly
providing us acll-2 seeds. This study was supported in part by a
grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences, a grant from "Research for the
Future" Program of the Japan Society for the Promotion of
Science (JSPS-RFTF96L00601), and a Grant-in-Aid for Scientific
Research on Priority Areas (A) "Molecular Mechanisms Controlling Multicellular Organization of Plant" (no. 10182206) from
the Ministry of Education, Science, Sports and Culture of Japan.
Inflorescence growth and senescence mutant
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(Received August 30, 1999; Accepted November 2, 1999)