Plant Physiol. (1998) 118: 1191-1201
Quantitative Control of Inflorescence Formation in
Impatiens balsamina1
Sylvie Pouteau2, *,
Fiona Tooke, and
Nicholas Battey
Department of Horticulture, Plant Science Laboratories, University
of Reading, Whiteknights, Reading, RG6 6AS, United Kingdom
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ABSTRACT |
We
analyzed the process of inflorescence formation in Impatiens
balsamina by studying the architecture of the plant under different photoperiod treatments. Floral reversion under noninductive conditions in this species is caused by the lack of persistence of the
induced state in the leaf. This can be used to control the amount of
inductive signal and to examine its quantitative influence on
morphological changes in the plant. The floral transition was
characterized by a continuum of variation at the level of meristem
identity, primordium initiation, and floral organ identity. This
continuum was enhanced during reversion, suggesting that the
establishment of a continuum partly reflects limiting amounts of
inductive signal exported from the leaf to the meristem. The transcription patterns of two homologs of genes involved in the control
of floral meristem identity, Imp-FLO and
Imp-FIM, were similar in terminal and axillary flowers
and may be associated with the continuum exhibited by I. balsamina. By analyzing the fate of axillary meristem primordia
initiated before and after the beginning of the inductive period, we
showed that de novo initiation of axillary meristem primordia by the
evoked meristem is not required and that primordia initiated before
evocation can adopt different fates, depending on the amount of
inductive signal. The influence of age and/or position on primordium
responsiveness to the inductive signal is discussed.
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INTRODUCTION |
The transition to flowering is characterized by dramatic changes
in plant morphology. These modifications commonly include changes in
leaf morphology and phyllotaxis, shortening of internodes, and flower
formation. Depending on the mode of growth and inflorescence formation,
flowering can occur at axillary positions on shoots or inflorescences
or as solitary flowers and may culminate in a terminal flower. Although
the subjects of inflorescence morphology and flowering physiology have
received much attention (Bernier, 1988
; Weberling, 1989; Bernier et
al., 1993), there have been few attempts to link these
disciplines.
Flowering can be triggered by a number of environmental stimuli,
including photoperiod and temperature. Photoperiod induction occurs in
the leaf and results in the formation of a mobile inductive signal.
Despite the broad variety of flowering responses to different stimuli,
the mechanisms that underlie the flowering process seem to be conserved
in different species. Graft transmission of flowering between species
with different photoperiod requirements suggests that the inductive
signal is universal, but its molecular nature has remained elusive.
Studies have revealed that the signal may be multifactorial (Bernier,
1988; Bernier et al., 1993).
The classical view of flowering physiology is that the inductive signal
is rapidly exported via the phloem sap to the shoot apical meristem,
which undergoes evocation (Evans, 1969; Zeevaart, 1976; Bernier, 1988;
McDaniel, 1992). The changes in the activity of evoked meristems cause
de novo initiation of flower primordia, but it is unclear how often
previously formed axillary meristem primordia are modified and whether
such modifications are mediated by the inductive signal from the leaf
directly or indirectly via the apical meristem. In plants with an
absolute photoperiod requirement it is possible to identify axillary
meristem primordia that are initiated before and after the beginning of
the inductive treatment and to analyze their fate in mature plants.
Furthermore, the manipulation of the level of inductive signal in the
plant during photoperiod treatments should provide information on the
mechanisms that control the progression to flowering and inflorescence
formation.
Impatiens balsamina is a very attractive model for the
analysis of the flowering process because it has an absolute
requirement for SD conditions for flowering, and flower reversion can
be obtained in a predictable way after transfer to LD conditions
(Battey and Lyndon, 1984, 1986, 1988, 1990; Pouteau et al., 1995, 1997,
1998). Both flower formation and reversion are characterized by a
continuum of changes in organ identity, and a large range of mosaic
organs is produced (Battey and Lyndon, 1988; Pouteau et al., 1998).
Following increasing amounts of induction in SD conditions, reversion
takes place at progressively later stages of flower development.
Reversion of the terminal flower correlates with the lack of
persistence of an induced state in the leaf (Pouteau et al., 1997).
Partial progression to flowering exhibited before return to leaf
formation can thus be considered to reflect the amount of inductive
signal exported from leaves before transfer to LD conditions.
In addition to the failure of leaves to become a permanent source of
inductive signal, flower reversion also implies that the terminal
meristem does not become committed to flowering in I. balsamina. Among the regulatory genes involved in flower
morphogenesis in snapdragon and Arabidopsis, a number are involved in
the specification of floral meristem identity. These include the
meristem identity genes FLO and LFY and their
mediators or coregulators, FIM and UFO, in
snapdragon and Arabidopsis, respectively (Coen et al., 1990; Weigel et
al., 1992; Simon et al., 1994; Ingram et al., 1995; Blázquez et
al., 1997; Lee et al., 1997). Analysis of the regulation of I. balsamina homologs of these genes (Imp-FLO and Imp-FIM, respectively) in the apical meristem shows a number
of similarities and differences (Pouteau et al., 1997, 1998).
Imp-FLO and Imp-FIM are transcribed during
vegetative growth, flowering, and reversion. However,
Imp-FIM specifically exhibits a new transcription pattern
during petal initiation and is not transcribed during the initiation of
reproductive organs, whereas Imp-FLO transcription is
apparently constitutive. However, it is unclear whether the new
transcription patterns of Imp-FLO and Imp-FIM are
specific to the apical meristem or whether the same transcription
patterns as those observed in snapdragon and Arabidopsis occur in
axillary meristems of I. balsamina.
We have analyzed the process of inflorescence formation in
I. balsamina by characterizing plant architecture
under continuous SD conditions and during reversion experiments carried
out after increasing periods of induction. Flowering over the whole
plant was characterized by a form continuum at three levels,
which was emphasized through the removal of the inductive signal by
transferring plants to noninductive, LD conditions.
The analysis of Imp-FLO and Imp-FIM transcription
in axillary flowers showed essentially no difference compared with
terminal flowers; the possible association between the regulation of
these two genes in I. balsamina and the gradual progression
to flowering is discussed. De novo initiation of axillary meristem
primordia by the evoked apical meristem is not required for flower
formation, and primordia initiated before apical meristem evocation
adopted different fates, depending on the amount of inductive signal
received. The degree of inflorescence development decreased basipetally in response to decreasing amounts of inductive signal. The youngest, uppermost axillary meristem primordia were most strongly induced in
response to SD conditions and their fate was least affected by transfer
to LD conditions. The influence of age and/or position on axillary
meristem responsiveness and the possible role of the apical meristem in
controlling flowering and inflorescence formation are discussed.
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MATERIALS AND METHODS |
Plant Material
We used an Impatiens balsamina cultivar (Dwarf Bush
Flowered) that is red-flowered and determinate and one that gives the most uniform reversion response. Plants were grown as previously described (Pouteau et al., 1997, 1998). Plant growth after sowing was
in LD conditions of 24 h at 21°C ± 1°C. At the top of
the plants on d 0, the total photon flux density was 260 to 280 µmol m
2 s1 during the day (8 h) and 5 µmol m2 s1
during the night (16 h). The compost was kept moist by the application of 200 mL of tap water per tray every day.
Photoperiod Treatments
Developmentally uniform plants with an average of nine primordia
were selected on d 0 (7 to 8 d after sowing and 10-11 d after imbibition). After d 0, flowering in SD conditions and flower reversion
after various periods of induction in SD conditions were
obtained as previously described (Pouteau et al., 1997). SD
conditions consisted of an 8-h period of illumination identical to that
applied for LD conditions, but complete darkness was maintained during
the 16-h night. No plant grown in continuous LD conditions developed
any floral features for at least 3 months.
Plants under different photoperiod treatments were randomly sampled at
different times for the preparation of material for in situ
hybridization assays. The number of nodes and primordia initiated by
the shoot apical meristem was determined in 10 plants at each sampling
time. Approximately 10 plants were grown until maturity to record the
characteristics at each node of organ identity, axillary shoot
identity, and internode elongation.
In experiments designed to analyze the influence of plant age on
flowering, plants were induced in SD conditions after seedling emergence (6 d before d 0), after d 0 (control), and 15 d after d
0. One-half of the plants was left under continuous SD conditions, and
the other half was transferred to LD conditions after 5 d of SD
conditions.
In Situ Hybridization
The methods for digoxigenin labeling of RNA probes, tissue
preparation, and in situ hybridization were as described by Bradley et
al. (1993). psep1-9 cut with HindIII and psep3-1 cut with
EcoRI were used as the templates for T7 RNA polymerase to
generate antisense and sense RNA probes of an Imp-FIM
fragment, respectively (Pouteau et al., 1998). pflo1 cut with
EcoRI and pflo7 cut with BamHI were used as
templates for T7 RNA polymerase to generate antisense and sense RNA
probes of an Imp-FLO fragment, respectively (Pouteau et
al., 1997). No signal was detected with sense RNA probes of Imp-FIM and Imp-FLO.
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RESULTS |
Continuum in Plant Architecture
Plant Architecture under Continuous SD Conditions
To identify the different axillary structures formed by the apical
meristem, plant architecture was analyzed under continuous inductive SD
conditions (Figs. 1 and 2). Plants formed a terminal inflorescence
(Fig. 1, A and B) consisting of a large
terminal flower and two or three solitary axillary flowers, each
subtended by a leaf (referred to as type-2 flowers). The organization
of the terminal flower was described previously (Battey and Lyndon, 1984; Pouteau et al., 1998). The lower type-2 flowers had a pedicel that was often partly or completely adnate to the main stem and were
subtended by normal leaves separated by internodes
(type-2p flowers). The upper type-2 flowers
lacked a pedicel and were borne in the axils of leaves that were not
separated by internodes (type-2np flowers). The
three nodes below the terminal inflorescence bore a leaf subtending an
axillary inflorescence. These structures were contracted inflorescences
consisting of a small number of flowers (two to five): these type-3
flowers were subtended by true bracts (i.e. leaves extremely reduced to
the size of small scales) and were not separated by internodes. The
five nodes below the lowermost axillary inflorescence bore a leaf that
subtended a flowering axillary shoot. The organization of these
structures recapitulated that of the main stem (Fig. 1B).
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| Figure 1.
Plant architecture under SD conditions. A,
Terminal inflorescence showing the terminal flower and two type-2
axillary flowers below. B, Diagram summarizing the main features of
plant architecture and the different types of axillary structures and
flowers. C, Rudimentary flower composed of only two unexpanded petals
and one filament (arrow). D, Rudimentary flower consisting of one
single filament. E, Rudimentary flower borne on an axillary shoot,
composed of one single sepal. F, Mosaic between a type-2 flower and an
axillary inflorescence showing a fasciated pedicel bearing three pods
but no bract. G, Mosaic between an axillary inflorescence and a
flowering axillary shoot showing a flower subtended by a leaf-bract
mosaic fused to the base of a shoot grown in the axil of a main stem
leaf. H, Same as G, but the pedicel of the flower at the base of the
shoot is adnate to the shoot stem. as, Stem of an axillary shoot; pe,
petiole of a main stem leaf; p, pedicel; st, main stem; s, sepal.
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| Figure 2.
Plant architecture during reversion. After initial
growth in LD conditions until d 0, plants were induced in SD conditions
for different times (4, 5, 6, 9, and 12 short days) and
transferred back to LD conditions. Control plants were grown after d 0 under continuous (Cont) SD conditions until maturity. Ten plants for
each treatment were analyzed and the number and identity of axillary
structures along the main stem were recorded: flowering axillary shoots
( ), axillary inflorescence/shoot mosaic structures
( ), axillary inflorescences of type-3 flowers ( ),
and type-2 flowers ( ). SEs varied between 0.13 and 0.50. The nodes initiated in LD conditions before transfer to SD
conditions are indicated below the d-0 mark, and those initiated after
transfer to SD conditions are indicated above the d-0 mark (Day 0).
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A continuum of changes in plant architecture could be observed at three
levels: (a) the formation of mosaic axillary structures that were
intermediate between the different classes of flowers, inflorescences,
and flowering shoots, as described above; (b) the progressive change of
axillary flower architecture along the main stem; and (c) the gradual
change in organ identity in the flowers.
Mosaic Axillary Structures
During flowering under continuous SD conditions and reversion
after transfer to LD conditions, mosaic axillary structures were
occasionally observed at the junctions between the zones giving rise to
axillary inflorescences and type-2 flowers. They usually consisted of
two or three flowers that were not subtended by bracts and had
partially fused pedicels (Fig. 1F). Mosaic axillary structures were
even more frequently observed at the junctions between the zones
corresponding to axillary inflorescences and flowering axillary shoots.
These structures, called mosaic shoots, corresponded to flowering
axillary shoots fused to the pedicel of a solitary flower subtended by
a bract or bract-like leaf (Fig. 1, G and H). Mosaic shoots were found
in 40% of the plants grown under continuous SD conditions.
Their frequency increased in response to reversion treatments: on
average, up to 1.9 nodes per plant exhibited mosaic shoots during
reversion after 5 d of SD conditions (Fig.
2; see below).
Gradual Change in Axillary Flower Architecture
Type-3 flower architecture and the gradual change in type-2 flower
architecture were analyzed in plants grown under continuous SD
conditions (Table I). Typical type-3
flowers were pentamerous and had 3 sepals, 10 petals, 5 stamens, and a
central pod comprised of 5 carpels. They were zygomorphic and usually
displayed 3 or 4 asymmetrical lateral petals, 5 symmetrical ventral
petals, and 1 large dorsal petal with a green tip and a green rib on
the abaxial side between the two lobes. The 3 sepals (2 lateral and 1 ventral) were spurred and similar in shape. Although most flowers had a total of 18 floral organs (excluding carpels), variations from one
flower to another were observed (extreme variants had 14 and 24 organs,
respectively) but were less pronounced than in the terminal flower
(Table I; Pouteau et al., 1998).
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Table I.
Flower architecture in the terminal inflorescence
Plants were grown under continuous SD conditions until maturity and
were dissected. Terminal and axillary flowers in 10 plants were
analyzed. This corresponded to 10 terminal flowers and 46 type 3 flowers (from axillary inflorescences), 12 type 2p flowers,
and 12 type 2np flowers. Sepals include regular and
modified sepals. True petals are fully expanded and
anthocyanin-pigmented petals. Staminate petals showed various degrees
of transformation into stamens. Asymmetrical petals were found mostly
in lateral position in the flowers. Data are ±SE.
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Type-2 flowers at gradually higher nodes showed a progressive reduction
in organ number and a decrease in sepal identity. Type-2p flowers had 1 sepal less but the same
number of petals and stamens compared with type-3 flowers.
Type-2np flowers had two sepals less, three or
four petals less, and one stamen less. Variation in organ numbers was
markedly higher than in type-3 flowers. In about 10% to 20% of the
plants, the most acropetal axillary structure corresponded to a
rudimentary structure that was often composed of one or two solitary
petals or a filament of unknown identity (Fig. 1, C and D).
Gradual Change in Floral Organ Identity
Mosaic or incomplete organs were commonly observed in type-3 and
type-2 flowers. In type-3 flowers an average of 0.4 of the 3.3 sepals
were modified and often had some petal features. An average of only 5.6 of the 10.0 petals were true petals (i.e. had 100% petal-pigmented
tissue; Pouteau et al. [1998]), and 4.4 petals displayed staminate
features. Approximately 30% of the pods had staminate features. Mosaic
organs were less frequent than in the terminal flower (Pouteau et al.,
1998).
Transcription of Imp-FIM and
Imp-FLO in Axillary Flowers
To determine whether the novel transcription pattern of
Imp-FIM during petal initiation and the constitutive
transcription of Imp-FLO during vegetative growth,
flowering, and reversion were specific to the apical meristem
(Pouteau et al., 1997, 1998), Imp-FIM and Imp-FLO
RNA patterns in type-2 axillary flowers were analyzed by in situ
hybridization.
The earliest expression of Imp-FIM in
type-2np flowers occurred after 8 d in SD
conditions, when the first petal primordium was initiated in the
terminal flower (Fig. 3A). This early
transcription corresponded to one single stripe of signal in the
meristem. Although no primordium was morphologically visible at this
stage, it is likely that it corresponded with the position of
initiation of the first sepal primordium. After this stage,
type-2np flowers developed in step with the
terminal flower. The Imp-FIM transcription pattern was
essentially identical in both types of flowers: it accumulated within
petal primordia but was absent from stamen primordia (Fig. 3, B and C).
Development of type-2p flowers was slightly
behind, but a similar pattern of Imp-FIM transcription was
observed within petal primordia (Fig. 3F).
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| Figure 3.
In situ hybridization analysis of
Imp-FIM and Imp-FLO transcription in
type-2 flowers. A to F, Imp-FIM transcription in a
terminal inflorescence after 8 d in SD conditions (A); in
type-2np flowers after 8 d in SD conditions (B),
17 d in SD conditions (C), and 5 SD + 15 LD (E); in a
vegetative axillary shoot after 8 d in LD conditions (D); and in
type-2p flowers fixed after 17 d in SD conditions (F).
G and H, Imp-FLO transcription in a terminal
inflorescence after 8 d in SD conditions (G) and in a vegetative
axillary shoot after 8d in LD conditions (H). Apical sections were
probed with digoxigenin-labeled Imp-FIM or
Imp-FLO antisense RNA and viewed under light-field
microscopy (the RNA signal is purple on a light-blue tissue
background). Leaf tissue and, more obviously, floral tissues remained
strongly pigmented after fixation and embedding due to the accumulation
of brown-stained granules. All photos were taken under a light-field
microscope with the same magnification factor. Scale bars = 100 µm. The terminal meristem (T) or stem tissue (st) are indicated when
visible to orient the sections. Arrowheads point to young axillary
floral meristems, and developing axillary flowers (Ax) are labeled.
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Plants transferred to LD conditions after 5 d in SD conditions had
the greatest axillary flower reversion; return to leaf formation
occurred after the production of a number of petals (see below; Fig.
5). In plants grown for 5 d in SD conditions and then 15 d in
LD conditions, Imp-FIM was transcribed mostly at the base of
the primordia in type-2np flowers (Fig. 3E). This
was similar to the pattern observed at the same stage in the terminal
meristem, which was initiating whorls of leaves at this time (Pouteau
et al., 1998). Therefore, transcription of Imp-FIM was
essentially identical in terminal and axillary flowers during flowering
and reversion. Transcription in vegetative meristems of axillary shoots
was as in the vegetative apical meristem (Fig. 3D).
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| Figure 5.
Reversion of type-2 axillary flowers. Reversion
treatments and the SD controls were as in Figures 2 and 4. Reversion of
type-2 flowers was analyzed in 10 plants for each treatment. The
frequencies of reverting () and nonreverting () flowers and of
rudimentary flowers with vegetative () or floral () features were
recorded from the lowermost to the uppermost node (left to right).
SEs varied from 0.10 to 0.35.
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Imp-FLO was transcribed in vegetative, flowering (Fig. 3, G
and H), and reverting axillary meristems, similar to the terminal meristem. After 8 d of SD conditions, a slight increase in
Imp-FLO transcript was observed in young axillary flower
primordia and in the terminal meristem (Fig. 3G; Pouteau et al., 1997).
Reversion Analysis of the Progression to Flowering
The progression to flowering under SD conditions can be described
by analyzing reversion in plants transferred to LD conditions after
different periods of induction (4-18 d) in SD conditions. The
progression to flowering in the terminal flower of I. balsamina during reversion was described previously (Pouteau et
al., 1997). We analyzed reversion in the remainder of the plant, below
the terminal flower.
Progression to Flowering in Axillary Meristems Produced after
Transfer to SD Conditions
In all reversion treatments and in the SD (flowering) control, the
first type-2 axillary flower was initiated in the axil of the youngest
primordium visible on d 0 (ninth leaf primordium; see ``Materials and Methods''; Figs. 2 and 4). Therefore,
the position of the first node bearing a type-2 flower was not
affected, even after inductive SD treatments as short as 4 d. Therefore, only primordia initiated on or after transfer
to SD conditions on d 0 were recruited to form the terminal
inflorescence.
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| Figure 4.
Progression to flowering in nodes initiated after
transfer to SD conditions. Control plants grown under continuous SD
conditions and plants transferred to LD conditions after different
periods of SD induction after initial growth in LD conditions until d 0 were as in Figure 2. Ten plants for each treatment were analyzed and
the lowermost nodes exhibiting different inflorescence traits were
recorded: Shaded box, type-2 flower;  , modified leaf; ×,
absence of internode above;  , leaf having petal pigmented sectors.
SEs varied between 0.18 and 0.52 and were higher for the
measures of the lowermost node not followed by an internode or with a
modified leaf after 4 SD + LD and the lowermost nodes with a leaf
containing petal pigment after 5 SD + LD and continuous SD (0.70, 1.10, 1.47, and 0.8, respectively). The areas corresponding to nodes
initiated under LD conditions before transfer to SD conditions (below
the d-0 mark [Day 0]) or after transfer from SD conditions are
shaded. The area corresponding to nodes initiated under SD conditions
is left blank.
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With an inductive SD treatment of 5 d or more, the total number of
type-2 flowers was the same as in the SD control (Fig. 2). However,
some or all of the axillary flowers reverted after an inductive SD
treatment of less than 12 d (Fig.
5). After 4 d in SD conditions
followed by LD conditions, about two-thirds of the type-2 structures
were virescent, with few floral features. Reversion treatments also
resulted in increased frequencies of rudimentary structures. These were
highest in treatments resulting in the highest reversion
responses (SD treatments of 4 and 5 d followed by LD treatment),
suggesting a link between them. In all SD treatments of less than
12 d, reversion of type-2 flowers was consistently observed in the
lowermost type-2 flower (Fig. 5). Therefore, the lowermost axillary
meristem of type-2 flowers either received a lower amount of inductive
signal or was less responsive to the inductive signal.
The transition from inflorescence features to terminal flower features
was gradual, and terminal flower features responded differently to the
amount of induction provided (Fig. 4). The repression of internode
elongation, the production of petal pigment in the appendages, and the
modification in shape and/or venation of the appendages required a
minimum SD treatment of 9, 6, and 5 d, respectively, to occur at
the same node level as in the SD control. The treatment of 5 d in
SD conditions followed by LD conditions was characterized by the most
severe uncoupling in the development of terminal flower features
compared with the SD control. After 4 d in SD conditions followed
by transfer to LD conditions, most floral features were repressed and
little morphological modification of the appendages occurred.
Progression to Flowering in Axillary Meristem Primordia Initiated
before Transfer to SD Conditions
Figure 2 shows how the fate of axillary meristems initiated before
transfer of plants to inductive SD conditions was strongly influenced
by the duration of the inductive treatment. After a SD treatment period
of 4 d, only a small number of axillary inflorescence/shoot mosaic
structures and no axillary inflorescences were formed. Only after
inductive SD treatments of 9 d or more did axillary inflorescences
develop, and these replaced the mosaic structures. Increasing the
duration of the inductive treatment therefore increased the extent of
inflorescence formation on the main stem in a basipetal direction.
Axillary shoots borne on the lowest five nodes of the main stem under
continuous SD conditions were identically organized. They formed a
terminal inflorescence consisting of a terminal flower and one solitary
type-2 flower below it (Fig. 1B). The three nodes below this terminal
inflorescence bore a leaf subtending an axillary inflorescence or an
axillary shoot. Approximately 10% of the main stem axillary shoots
displayed rudimentary structures in their terminal inflorescence. In
contrast to those in the main stem terminal inflorescence, these
rudimentary structures usually comprised one or two sepals and, less
frequently, a filament (Fig. 1E; Table
II).
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Table II.
Organization of flowering axillary shoots
Plants were grown under SD conditions for different periods (13, 15, and 18 d) and transferred to LD conditions. Control plants were
grown under continuous SD conditions until maturity. Flowering axillary
shoots (38, 39, and 41, respectively) from 10 plants were dissected for
the 13 SD + LD, 15 SD + LD, and 18 SD + LD treatments.
Sixty-nine flowering axillary shoots from 14 control SD plants were
analyzed. The number and type of axillary structures borne on each
shoot were recorded (axillary shoots and inflorescences and type-2
flowers). Rudimentary type-2 flowers corresponded in most cases to one
or two sepals and less frequently to a filament. The percentage of
reversion in the terminal flowers of axillary shoots was recorded in
36, 30, 31, and 52 axillary shoots in the 13 SD + LD, 15 SD + LD, 18 SD + LD, and control treatments, respectively. Data are
±SE.
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Analysis of axillary shoot architecture during reversion experiments
showed an increase in the number of type-2 flowers compared with those
treated with continuous SD conditions, even after SD inductions as long
as 18 d (Table II). This increase was more pronounced in the
uppermost axillary shoots, where up to two more type-2 flowers were
formed in the plants given 15 d of SD treatment followed by LD
treatment than in plants given continuous SD treatment. There was no
detectable increase in the number of type-2 flowers on axillary shoots
at the two lowest nodes. A large part of this increase resulted from an
increase in the number of rudimentary structures, up to about one
rudimentary structure per branch after 15 d of SD treatment
followed by LD treatment (Table II). Reversion of terminal flowers on
axillary shoots was high after 18 d of SD treatment followed by LD
treatment (65%) and increased to 90% after 15 d of SD treatment
followed by LD treatment (Table II). In contrast, no terminal flower on
the main stem reverted after 18 d of SD treatment followed by LD
treatment, and only 10% of them reverted after 15 d of SD
treatment followed by LD treatment (Pouteau et al., 1997).
Influence of Plant Age on the Progression to Flowering
To determine the effects of plant age on the progression to
flowering, induction and reversion after 5 d of SD treatment were carried out at emergence of the seedlings (d 6) and 15 d after d 0 and compared with d-0 controls (Fig. 6).
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| Figure 6.
Influence of plant age on the progression to
flowering. Flowering under continuous SD conditions (F) and reversion
after 5 SD + LD (R) were carried out at three stages after initial
growth in LD conditions: at emergence of the seedlings 6 d before
d 0 (D-6), on d 0 (Day 0), and on d 15 (D+15). The areas corresponding
to nodes initiated under LD conditions are shaded, and the areas
corresponding to nodes initiated under SD conditions are left blank.
The number of nodes initiated in LD conditions before transfer into SD
conditions fall below the "Day  6," "Day 0," and "Day 15"
marks, respectively, in the three treatments. The number of nodes
initiated at emergence of the seedling was not recorded; therefore, an
estimate is given. Ten plants for each treatment were analyzed, and the
number and identity of axillary structures along the main stem were
recorded. Symbols are as in Figure 2. SEs varied between
0.16 and 0.40. Reversion in the terminal flower was recorded using the
reversion scale described in previous work (Battey and Lyndon, 1984;
Pouteau et al., 1997). This scale describes the degree of flower
development before return to leaf initiation. It ranges from R0 (no
flower development) to R8 (carpels). Intermediate reversion types
include R1 (virescent axillary structures), R3 (repression of internode
elongation), R4 (modified venation, petal pigment), R5 (petals), and
R6/7 (stamens). F, Nonreverting flower. The frequency of reversion in
type-2 flowers was recorded.
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In the d-6 experiments, the number of type-2 flowers initiated under
continuous SD conditions was similar to the d-0 control, but fewer
axillary inflorescences and mosaic shoots were formed. The amount of
induction during the 5 d of SD treatment after emergence was less
compared with the d-0 control, since transfer to LD conditions after
this time resulted in drastic reduction of floral features in the whole
plant. Reversion in the terminal flower occurred significantly earlier
than in the d-0 controls, and 40% of the plants had few or no
flowering features. No axillary inflorescence or mosaic shoot was
formed, and half as many type-2 flowers were formed, all of which
reverted.
In contrast, the inductive effect of 5 d of SD treatment when
given 15 d after d 0 was markedly higher than in the d-0 control: reversion in the terminal flower occurred later, mostly after stamen
formation, the different classes of axillary structures were mostly
unaffected, and only 9% of type-2 flowers reverted. Under continuous
SD conditions from d 15, type-2 flowers and axillary inflorescences and
mosaic shoots were all derived from primordia initiated before transfer
to SD conditions. The total number of type-2 flowers was slightly
increased, and there were about 3 times more axillary inflorescences
than in the d-0 controls. These observations suggest that the plant
becomes more responsive to SD induction as it ages. Also, the fate of
axillary meristems remains uncommitted until late and can be altered if
a sufficient amount of induction is provided, possibly through an
increased number of receptive leaves or through increased competence of the meristem.
| |
DISCUSSION |
Form Continuum in I. balsamina
The analysis of inflorescence architecture in I. balsamina shows that flowering progresses as a continuum at three
levels: (a) meristem identity, in which the formation of mosaic
structures is observed at the junctions between the zones marked by
axillary flowering shoots, axillary inflorescences, and flowers of the terminal inflorescence; (b) primordium initiation, in which successive axillary flowers exhibit a gradual reduction in the total number of
floral organs, with extreme reduction to one or two organs in some of
the uppermost flowers and a reduction in sepal identity; and (c) organ
identity, which changes gradually in successive organs produced in
terminal and axillary flowers and is accompanied by the formation of
mosaic organs (Pouteau et al., 1998; Tooke et al., 1998; this work).
Progressive changes in plant morphology are also observed in other
species during the transition from vegetative to floral development.
This is often characterized by heteroblasty in successive leaves, which
show gradual changes in morphology (Poethig, 1997), or by gradual
changes in organ identity adopted by successive primordia, such as in
the Nymphaeaceae family (Sporne, 1974). Furthermore, mosaic flowering
shoots have been described in a number of mustard species including
Arabidopsis (Hempel and Feldman, 1995; Hempel, 1996), in which
flowering mutants often exhibit a progressively weaker mutant phenotype
in successive flowers along the main axis (Haughn et al., 1995).
The concept of continuum morphology can be applied to interpret the
form continuum exhibited by I. balsamina and other species during the transition from vegetative to floral development. According to continuum morphology, as opposed to classical morphology, plant organs and structures are not sharply delimited from each other but
instead form a continuum. The plant itself constitutes a morphological unit in which various morphological subunits are successively integrated and are continuously modified throughout the life of the
plant (Sattler, 1996; Sattler and Rutishauser, 1997). The form
continuum exhibited during flowering in I. balsamina
suggests the quantitative nature of underlying developmental changes.
Influence of the Inductive Signal on the Form Continuum
The evidence suggests that this continuous variation in form
reflects the amount and/or translocation rate of the inductive signal
from the leaf. Removal of the inductive signal in I. balsamina results in reversion and can cause an increase in the
form continuum at all three levels mentioned above (Pouteau et al.,
1998; Tooke et al., 1998; this work). Increased developmental
plasticity during reversion is also illustrated by the uncoupling of
terminal inflorescence traits such as the formation of axillary
flowers, the suppression of internode elongation, and modifications in
leaf morphology.
Reversion in a number of other species under suboptimal or
noninductive conditions can also reveal more progressive changes than
those observed under continuous inductive conditions (Battey and
Lyndon, 1990). Noninductive conditions can also cause a more pronounced
form continuum in nonreverting species such as Arabidopsis and
snapdragon. Arabidopsis plants grown under noninductive
conditions exhibit a more gradual transition from rosette leaves to
cauline leaves and to leaf suppression and increased severity in
flowering mutant phenotypes (Haughn et al., 1995; Okamuro et al., 1996, 1997; Mizukami and Ma, 1997). In snapdragon transfer experiments from
inductive to noninductive conditions resulted in more gradual changes
and uncoupling of inflorescence features (Bradley et al., 1996).
Therefore, a sufficient quantity of inductive signal may be required in
most species to allow rapid progression to flowering and to mask the
gradual nature of developmental changes that underlie this transition.
Role of Meristem Identity Genes in the Progression to Flowering
The activation of genes involved in the control of floral meristem
identity has been shown to participate in rapid progression to
flowering in Arabidopsis (Haughn et al., 1995; Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995). The I. balsamina homologs
of two meristem identity genes, Imp-FLO and
Imp-FIM, exhibit a number of differences in their
transcription patterns in the apical meristem of I. balsamina compared with the patterns of their orthologs observed
in Arabidopsis and snapdragon axillary meristems (Pouteau et al., 1997,
1998). Here we show that Imp-FLO and Imp-FIM
transcription patterns are essentially the same in terminal and
axillary flowers of I. balsamina. Therefore, the differences
observed from other species are not specific to the terminal flower.
These differences are also observed in the apical meristem of a
nonreverting line of I. balsamina in which the progression
to flowering is gradual, as in the reverting line used in this work
(Pouteau et al., 1998; F. Tooke and N.H. Battey, unpublished
data). Nonreversion in this line results from the persistence of an
induced state in the leaf, and reversion can be obtained by removing
the induced leaves (Tooke et al., 1998). It is therefore possible that
the specific pattern of Imp-FLO and Imp-FIM
transcription in I. balsamina is associated with the lack of
commitment of the meristem in this species.
Quantitative Control of Inflorescence Formation
The apical meristem is usually considered to be the main recipient
of the inductive signal exported from the leaf. As a result of
evocation, the apical meristem is expected to act as the mediator of
flowering in the plant (Bernier, 1988, 1997). The prevailing sequential
interpretation of flowering has led to the postulate that floral
axillary structures are initiated de novo by the apical meristem after
the onset of evocation. For example, in white mustard the initiation of
the first flowers occurs 60 h after the beginning of the inductive
LD conditions (Bernier, 1997). In Arabidopsis the existence of three
different phases of development has been suggested previously based on
the observation of three distinct types of plant morphological units
marked, respectively, by rosette leaves, axillary flowering
shoot/cauline leaves, and flowers (Schultz and Haughn, 1991; Huala and
Sussex, 1992; Shannon and Meeks-Wagner, 1993). However, by determining
when primordia are initiated relative to the beginning of the inductive
treatment, it has been shown that the shoot apical meristem can cease
producing leaf primordia and begin to produce flowers during the first
inductive photoperiod cycle (Hempel and Feldman, 1994). This led to the
conclusion that there are only two phases of development in
Arabidopsis, a vegetative phase and a reproductive phase, the latter
being characterized by de novo initiation of flower primordia.
We show that axillary flower production in I. balsamina does
not require de novo initiation of primordia by the apical meristem. Although during the standard inductive treatment from d 0, the lowermost axillary flower was produced in the axil of the youngest primordium morphologically detectable at the time of transfer to SD
conditions, after induction at a later stage (i.e. from d 15) all
axillary flowers were derived from primordia initiated before the
beginning of the inductive treatment, and transfer to LD conditions
after 5 d of SD conditions was less effective in promoting
reversion. This stronger induction response could reflect increased
competence of the plant to respond to the inductive signal, but,
because more leaves are present, a more likely explanation is that this
reflects a higher amount of inductive signal.
Because it results from the lack of persistence of the induced state of
the leaf (Pouteau et al., 1997; Tooke et al., 1998), reversion in
I. balsamina provides a means to analyze the quantitative influence of the inductive signal on inflorescence development. The
gradual increase in floral features exhibited by mature plants after
progressively longer periods of induction in SD conditions is expected
to reflect the increase in the number of induced leaves and in the
amount of inductive signal exported by them. Analysis of fate changes
in axillary meristem primordia initiated before transfer to inductive
SD conditions on d 0 shows that these primordia can generate axillary
shoots, mosaic axillary shoots, or axillary inflorescences, depending
on the amount of inductive signal in the plant. We conclude that the
progression of flowering in the plant depends on the amount of
inductive signal, which is influenced by external inductive conditions
and plant age. Therefore, the apparent requirement for de novo
initiation of axillary meristem primordia in other plants, such as
white mustard and Arabidopsis (Hempel and Feldman, 1994; Bernier,
1997), may be fortuitous and result from insufficient inductive signal
under the experimental conditions.
Basipetal Progression of Inflorescence Formation
Although its specific cell-partitioning function is not required
for flower initiation, it is possible that the evoked apical meristem
acts as the controller of flowering by being the main recipient of the
inductive signal exported from the leaf. However, it is
unclear whether the inductive signal can be directly exported into
developing axillary meristems. Hempel and Feldman (1995) found in
Arabidopsis that the sides of mosaic flowering shoots farthest from the
apical meristem are specified as "flowers" and concluded from this
observation that the inductive signal coming from the leaf can directly
induce primordia to develop as flowers. However, it is possible that
the side farthest from the apical meristem is the most responsive to
the inductive signal coming from the leaf, either directly or via the
apical meristem.
Analysis of fate changes after progressively longer periods of
induction in axillary meristem primordia initiated before and during
the inductive treatment shows that the development of floral traits is
greatest in uppermost primordia and decreases in progressively lower
primordia. One interpretation could be that the position of primordia
relative to the apical meristem is important. Floral conversion may be
more efficient in the uppermost primordia than in lower primordia
because they are nearest the apical meristem. This would imply that a
direct influence of the inductive signal from the leaf is not essential
and that this influence is mostly mediated by the apical meristem.
According to this interpretation, the apical meristem would act as the
main recipient of the inductive signal from the leaf and would
therefore control the specification of axillary flowers and
inflorescences, possibly through the production of a secondary signal.
Another interpretation could be that the age of axillary meristem
primordia rather than their position relative to the apical meristem is
important. Uppermost primordia could be the most responsive to the
inductive signal, irrespective of its acting directly from the leaf or
via the apical meristem, because these primordia are the youngest at
the beginning of the inductive treatment. However, a difficulty for
type-2 flower primordia that are initiated after transfer to SD
conditions is that the upper ones are more responsive to the inductive
signal than the primordia below, although the former must be induced
for a shorter period than the latter. One explanation could be that
upper type-2 flower primordia are influenced by higher amounts of
inductive signal at an earlier stage. Alternatively, although they
cannot be detected at a morphological level at the time of transfer to
SD conditions, these primordia could be already partitioned as cell
sectors. In any case, primordia initiated after the transfer to SD
conditions and the youngest primordia initiated before transfer to SD
conditions correspond to anlagen, which differentiate only later into
leaf/internode/axillary structure units. It would be interesting to
determine to what extent the fate of axillary structures is influenced
by the previous history of their anlagen.
In summary, inflorescence architecture in I. balsamina can
be explained by the response of axillary meristem primordia to the
quantity of inductive signal, a response that is conditioned by the age
and/or position of the primordia and allows undifferentiated axillary
meristem primordia initiated before evocation to adopt different fates.
This developmental plasticity results in various combinations of
vegetative and floral characters. Our interpretation is that vegetative
and reproductive phases are not separate and antagonistic but
interpenetrate each other to varying extents depending on the quantity
of inductive signal.
| |
FOOTNOTES |
1
This work was funded by the Biotechnology and
Biological Science Research Council Cell Molecular Biology Initiative
(grant no. AT45/559 to F.T.). S.P. was supported by the Institut
National de la Recherche Agronomique, Versailles, France.
2
Present address: Laboratoire de Biologie
Cellulaire, Institut National de la Recherche Agronomique, Route de
Saint-Cyr, F78026 Versailles cedex, France.
*
Corresponding author; e-mail pouteau{at}versailles.inra.fr; fax
33-1-30-83-30-99.
Received June 30, 1998;
accepted August 28, 1998.
| |
ABBREVIATIONS |
Abbreviations:
LD, long-day.
SD, short-day.
| |
ACKNOWLEDGMENTS |
We are grateful to Dr Enrico Coen and members of his laboratory
and to members of the laboratory of N.B. for their support and
encouragement.
| |
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Abstract
Introduction