1 Institute of Molecular & Cell Biology, 30 Medical Drive, Singapore
117609
2 Department of Biological Sciences, National University of Singapore, Singapore
117546
3 Institute of Plant Physiology & Ecology, 300 Fenglin Road, Shanghai
200032, China
4 John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
Authors for correspondence (e-mail:
dluo{at}sibs.ac.cn,
harberd{at}bbsrc.ac.uk
and
pengjr{at}imcb.a-star.edu.sg)
Accepted 18 November 2003
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SUMMARY |
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Key words: Gibberellin, DELLA proteins, Stamen development, Floral development
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Introduction |
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The histology of anther development (microsporogenesis) is well documented
(Goldberg et al., 1993;
McCormick, 1993
;
Sanders et al., 1999
). In
addition, the study of mutants exhibiting stage-specific defects in
microsporogenesis and pollen release has further advanced our understanding of
the process (Preuss et al.,
1993
; Sanders et al.,
1999
; Park and Twell,
2001
; Azumi et al.,
2002
; Steiner-Lange et al.,
2003
; Unte et al.,
2003
). Although little is known about how GA controls stamen
filament elongation, anther development or microsporogenesis, there have been
previous suggestions that GA-signalling components may modulate these
processes. For example, overexpression of SPY (an Arabidopsis
GA-signalling component) (Jacobsen et al.,
1996
) inhibits post-meiotic anther development in petunia
(Izhaki et al., 2002
).
Furthermore, transgenic expression of wild-type or mutant forms of GAI
(another Arabidopsis GA-signalling component, see below) can retard
stamen elongation and induce male-sterility in tobacco and
Arabidopsis (Huang et al.,
2003
; Hynes et al.,
2003
). By contrast, infertility caused by impaired floral
development is also a characteristic of mutants lacking the rice or barley
DELLA proteins SLR1 or SLN1 (Ikeda et al.,
2001
; Chandler et al.,
2002
). Despite these various reports, the mechanism via which GA
regulates petal, stamen and anther development remained unclear.
Recent advances have enabled the identification of a family of proteins
homologous to Arabidopsis GAI and RGA
(Peng et al., 1997;
Silverstone et al., 1998
) that
are crucial for the regulation of plant stem elongation growth in response to
GA (Peng et al., 1999
;
Ikeda et al., 2001
;
Boss and Thomas, 2002
;
Chandler et al., 2002
). These
proteins belong to the DELLA family (Fleck et al., 2002), a subfamily of the
GRAS family of putative transcriptional regulators
(Pysh et al., 1999
;
Richards et al., 2000
). The
Arabidopsis genome encodes five distinct DELLA proteins
(Lee et al., 2002
). Genetic
suppression studies have shown that GAI and RGA functions overlap in the
repression of plant stem growth (Dill and
Sun, 2001
; King et al.,
2001
). Further studies showed that while RGL2 controls seed
germination (Lee et al.,
2002
), RGL1 may control stem elongation as well as seed
germination (Wen and Chang,
2002
). Although GAI, RGA, RGL1 and RGL2 are all
expressed in developing inflorescences
(Lee et al., 2002
), no obvious
suppression of ga1-3 floral phenotype was observed in ga1-3
mutants lacking GAI, RGA, GAI and RGA, or RGL2
(Dill and Sun, 2001
;
King et al., 2001
;
Lee et al., 2002
). However, a
transgenic RGL1 loss-of-function line was resistant to the arrest of
floral organ development induced by paclobutrazol (PAC, an inhibitor of GA
biosynthesis) (Wen and Chang,
2002
), suggesting that RGL1 might play a role in regulating floral
development. These observations underscore the importance of determining
systematically the respective roles of the various DELLA proteins in
GA-mediated regulation of Arabidopsis petal and stamen
development.
In this report, we describe experiments addressing two questions. First, we characterized ga1-3 floral development to determine at which stage of stamen/anther development GA-deficiency causes developmental arrest. Second, we used novel combinations of loss-of-function mutations to determine if DELLA proteins are repressors of stamen filament elongation and microsporogenesis. Our results show that GA is crucial both for cell elongation during stamen elongation and for the developmental progression from microspore to mature pollen grain during pollen development. We also show that the DELLA proteins RGA, RGL1 and RGL2 work together to repress stamen and anther development in GA-deficient plants.
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Materials and methods |
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Histology and in situ hybridization
For scanning electron microscopy, individual flower buds from fresh
wild-type or mutant inflorescences were dissected; outer organs were removed
using stainless steel needles. Buds were attached to a mounting plate, plunged
into liquid nitrogen, quickly transferred to a specimen chamber and scanned at
10 KV (JSM-5310LV, JEOL, Japan). Pollen grains were mounted on scanning
electron microscopy stubs and coated with gold using previously described
techniques (Bozzola and Russell,
1999). For anther sectioning, fresh inflorescences were fixed in
formalin-acetic acid-alcohol (FAA) fixative buffer at 4°C overnight
followed by dehydration steps and subsequent embedding in Jung Historesin
(Leica). Sections (2.5 µm) were made using a Leica RM 2055 microtome and
stained with 0.25% Toluidine Blue O (Sigma). DAPI staining of pollen grain
nuclei was performed as described (Chen and
McCormick, 1996
) and pollen numbers were counted under a
microscope (Leica DM RXA2) with 40x or 20x objectives. Color
photos were taken using a Spot Insight QE digital camera (Diagnostic
Instruments). The ATA7 and SDS antisense and sense probes
were synthesized from the pMC1577 and pMC2317 plasmids, respectively
(Zhao et al., 2002
) and in
situ hybridisation was performed as described previously
(Luo et al., 1996
). Callose
staining and chromosome spread analysis of meiotic stages were as described
(Regan and Moffatt, 1990
;
Ross et al., 1996
).
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Results |
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The surface structure of the mature pollen grains released by wild-type plants was compared with that of immature pollen grains manually dissected from ga1-3 anther locules. All wild-type pollen grains were oval shaped with long indented lines. Very few oval shaped pollen grains were observed in ga1-3. Instead, in most cases, the immature pollen grains from ga1-3 plants were spherical in shape (Fig. 2A). Wild-type and ga1-3 anthers were dissected and stained with DAPI. As expected, the mature pollen grains from wild-type plants were tricellular, and contained three nuclei (Fig. 2B). However, in most cases (see Discussion), fewer than 10% of the developing grains examined in ga1-3 pollen sacs were found to be bicellular/tricellular (Fig. 2B,C). In fact, about 48% of ga1-3 pollen grains contained only a single nucleus and 46% had no nucleus. Clearly, ga1-3 fails to produce mature pollen, and this probably results from an arrest or impairment in pollen development prior to or during pollen mitosis in ga1-3 (Fig. 2C).
|
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First, we studied ga1-3 plants lacking single DELLA proteins.
Absence of RGL1, RGL2 or GAI alone had little obvious effect on the phenotype
of ga1-3, while absence of RGA alone partially suppressed the stem
elongation phenotype of ga1-3
(Silverstone et al., 1998)
(Fig. 5A). However, absence of
RGA alone did not restore normal floral development or fertility to
ga1-3 (Fig. 5C).
|
For the most part, the pair-wise DELLA absence combinations failed to
confer normal flower development on ga1-3
(Dill and Sun, 2001;
King et al., 2001
). All of
these lines produced flower buds, but the buds failed to open and exhibited
the arrested petal and stamen growth characteristic of ga1-3
(Fig. 7A). However, some flower
opening was observed in the late maturity of two of the pair-wise DELLA
absence combination lines. Although flowers of 40-day old ga1-3
plants lacking RGL2 and RGA were sterile, at
50 days and older these
plants produced flowers that opened and were able to set seed
(Fig. 5C). In addition, the
flowers of late maturity ga1-3 plants lacking GAI and RGA (
55
days old; data not shown) sometimes opened, but these flowers were almost
always sterile.
|
|
ga1-3 plants lacking RGL2, GAI and RGA were also taller at maturity than control lines lacking GAI and RGA alone (Fig. 6A,B,D). In contrast to what was seen with lack of RGL1, lack of RGL2 (in ga1-3 plants lacking RGL2, GAI and RGA) partially restored petal and stamen development to ga1-3 plants lacking GAI and RGA, making this line partially fertile (Fig. 1B and Fig. 7A,B).
In summary, the results described in this section indicate that GA-regulation of Arabidopsis petal and stamen elongation is mediated via RGL1, RGL2 and RGA, with RGL2 and RGA playing the predominant roles.
Absence of RGA, RGL2, RGL1 and GAI leads to GA-independent plant growth
Finally, we analysed ga1-3 plants lacking RGL1, RGL2, GAI and RGA.
We found that this mutant line bolted and flowered earlier than wild type both
in long (LD) (Fig. 6A,C) and
short (SD) days (Fig. 6C).
Furthermore, ga1-3 plants lacking RGL1, RGL2, GAI and RGA were taller
than the wild-type control (Fig.
6B,D). In addition, combined absence of RGL1, RGL2, GAI and RGA
suppressed the effects of ga1-3 on petal and stamen development. The
flowers of ga1-3 plants lacking RGL1, RGL2, GAI and RGA exhibited
fully extended stamens and petals (Fig.
7A). Anther development proceeded to completion, resulting in
flowers that were fertile and set seeds in both LD
(Fig. 7B) and SD (data not
shown).
We next analyzed stamen filament growth in ga1-3 plants lacking RGL1, RGL2, GAI and RGA and found that these filaments were slightly longer than those of the wild-type control (Fig. 1A,B). In fact, removing only RGL1, RGL2 and RGA from ga1-3 also resulted in filament lengths longer than that of wild type (Fig. 1A,B). SEM of stamen filament epidermal cells indicated that restoration of stamen filament length in ga1-3 mutant plants lacking RGL1, RGL2, GAI and RGA was due to an increase in cell length (Fig. 1C) as opposed to an increase in cell number (Fig. 1D), a difference similar to what was previously observed between wild-type and ga1-3 stamen filaments. Thus, the elongation of stamen filaments becomes GA independent when all four DELLA proteins are removed.
SEM analysis of pollen grains from ga1-3 plants lacking RGA, RGL2
and RGL1 showed that their surface structure was indistinguishable from those
of wild type (Fig. 2A), being
oval-shaped with long indented lines. However, pollen grains from
ga1-3 plants lacking RGL1, RGL2, GAI and RGA were substantially
different from those of wild type, mostly having a wrinkled appearance, and
sometimes being markedly deformed (Fig.
2A). Although ga1-3 plants lacking RGL1, RGL2 and GAI had
hugely different stem elongation phenotypes to ga1-3 plants lacking
RGL1, GAI and RGA (Fig. 6A,B),
the floral phenotypes of these two lines are very similar and both lines had
30% tricellular pollen (Fig.
2C). This suggests that microsporogenesis is partially restored in
these two lines. Lack of both RGL2 and RGA had a greater effect on
microsporogenesis, as
60% or
80% of pollen grains were found to be
tricellular in ga1-3 plants lacking RGL2, GAI and RGA or RGL1, RGL2
and RGA respectively (Fig.
2B,C). These results indicate that RGA and RGL2 play important
roles in the repression of microsporogenesis in Arabidopsis, and that
GA regulates microsporogenesis by overcoming the repressing effects of RGA and
RGL2.
Transverse sectioning showed that ga1-3 plants lacking RGL1, RGL2 and RGA or ga1-3 plants lacking RGL1, RGL2, GAI and RGA both achieved complete microsporogenesis (Fig. 3). However, although no obvious differences were observed between wild-type and ga1-3 plants lacking RGL1, RGL2 and RGA, we often observed that one or two of the four locules of the anthers of ga1-3 plants lacking RGL1, RGL2, GAI and RGA were aborted (data not shown).
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Discussion |
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Previous studies of Arabidopsis DELLA function have involved
phenotypic comparisons of GA-deficient (ga1-3) plants with
GA-deficient plants lacking GAI, RGA, RGL1, or RGL2 or a limited range of
combinations of these factors (Dill and
Sun, 2001; King et al.,
2001
; Lee et al.,
2002
). In this paper, we described the effects of a more
comprehensive set of DELLA-lack combinations, focussing especially on floral
development.
Flowering consists of three distinct phases: floral initiation (in which
the vegetative meristem is transformed into an inflorescence meristem), floral
organ initiation and floral organ growth. As shown previously, lack of GAI and
RGA substantially suppresses the effect of the ga1-3 mutation on
flowering time (a measure of time of floral initiation) in SD
(Dill and Sun, 2001). We have
shown that an additional lack of RGL2 or of both RGL1 and RGL2 further
advances the flowering time (in both LD and SD) of ga1-3 plants
lacking GAI and RGA. However, the magnitude of this further advance is
relatively small compared with that initially caused by lack of both GAI and
RGA. Thus, GAI and RGA play the predominant role in regulating flowering time
in the GA-signalling floral promotive pathway
(Simpson and Dean, 2002
), with
only small contributions from RGL1 and RGL2.
By contrast, RGL1, RGL2 and RGA play key roles in floral organ development.
The temporal coordination of the development of individual floral organs is
essential for floral function. For example, at around the time that the pollen
matures and is dehisced from the anther, the stamen filaments of flowers of
self-fertilizing species such as Arabidopsis elongate and bring the
pollen into contact with the stigmatic papillae
(Smyth et al., 1990;
Bowman, 1994
). We showed that
the relatively short stamen filaments of ga1-3 flowers result from an
arrest of cell elongation rather division and that combined lack of RGL1,
RGL2, GAI and RGA restored stamen filament cell elongation in ga1-3
plants. We also showed that, in general, microspores do not proceed to the
formation of mature pollen in ga1-3 anthers, that microspore
development is possibly arrested prior to pollen mitosis in ga1-3,
and that tapetal development is perturbed in ga1-3. Whether the
effect of ga1-3 on pollen mitosis is a secondary effect of arrested
tapetal development, or is independent of the effect on tapetal development is
at present unclear. In addition, we occasionally observed ga1-3
flower buds containing a significant number of tricellular pollen grains.
Further investigation is needed to find out if this is a true reflection of
the ga1-3 developmental process or is caused by other unknown
environmental cues. Lack of RGL1, RGL2, GAI and RGA proteins restored
microsporogenesis in ga1-3 plants. Further genetic analysis enabled
us to identify RGL2, RGA and RGL1 as the key GA-response regulators
controlling stamen filament length and microsporogenesis. Interestingly,
pollen grains from ga1-3 plants lacking GAI, RGL1, RGL2 and RGA,
although tricellular and viable, are deformed when compared with the
wild-type-appearing pollen grains from ga1-3 plants lacking RGL1,
RGL2 and RGA. Perhaps absence of all four DELLA proteins activates the GA
pathway to such high levels that pollen wall materials are overproduced,
resulting in abnormal pollen morphology.
Previous developmental genetic analyses showed that the
Arabidopsis DELLA proteins GAI and RGA act as repressors of stem
elongation and that GA exerts its promotive effects on stem growth by
overcoming the effects of GAI and RGA (Dill
and Sun, 2001; King et al.,
2001
). These observations, and additional observations on the
behaviour of DELLA proteins in other species, have been incorporated into a
general `release of restraint' model, which envisages DELLA proteins as
general agents of restraint of plant organ growth, and GA as a means of
overcoming that restraint (Peng et al.,
1997
; King et al.,
2001
; Richards et al.,
2001
; Harberd,
2003
). However, the initial experiments (which examined the effect
of lack of Arabidopsis GAI and RGA) showed that although stem
elongation could be explained in terms of the `release of restraint' model,
other aspects of growth and development which were known to be GA regulated
(in particular seed germination and floral organ growth) could not
(Dill and Sun, 2001
;
King et al., 2001
). It
therefore remained possible that some other, entirely different, mechanism was
responsible for the GA-mediated regulation of seed germination and floral
organ growth.
Recently, it has been reported that the GA-promotion of
Arabidopsis seed germination can be explained in terms of a
GA-mediated release of the restraint upon germination imposed by RGL2
(Lee et al., 2002) or RGL1
(Wen and Chang, 2002
). The
results in this present paper show for the first time that the GA-regulation
of floral organ development is also DELLA-mediated. However, different
combinations of DELLA proteins are key to floral organ development (RGA, RGL1,
RGL2), seed germination (RGL2 and RGL1) and stem elongation (RGA, GAI). The
three key aspects of the ga1-3 mutant phenotype (dwarfism, inhibition
of seed germination, retarded floral organ development) can now be explained:
the lack of GA in this mutant causes a failure to overcome the repressive
effects of the DELLA protein combinations that are specific to each particular
phenotypic aspect. As a consequence, the `release of DELLA restraint'
hypothesis can now be considered to be a viable model with which to understand
GA responses in general. One possible explanation for how different DELLA
combinations control different developmental processes (e.g. seed germination
versus stem elongation versus stamen development) is that individual DELLA
proteins have different temporal and spatial expression patterns. For example,
GAI and RGA are ubiquitously expressed in all plant tissues,
whereas RGL1 and RGL2 transcripts are relatively enriched in
the inflorescence (Silverstone et al.,
1998
; Lee et al.,
2002
; Wen and Chang,
2002
). In situ hybridisation showed that RGL1 is highly
expressed in the stamen primordium (Wen
and Chang, 2002
); however examination of an RGL2
promoter-GUS fusion line showed that RGL2 transcripts are also
enriched in the stamen (Lee et al.,
2002
). The expression patterns of RGL1 and RGL2
are therefore consistent with our current observation that RGL1 and RGL2 are
both important for stamen development. It is possible that RGL3 also plays an
important role in various aspects of GA-mediated developmental regulation.
Determination of the relative role of RGL3 awaits characterization of
Arabidopsis mutants lacking the functional RGL3 allele.
The nature of the arrest in flower development conferred by ga1-3 (and restored by lack of RGL1, RGL2 and RGA) is particularly interesting. Our results identify a relatively distinct developmental stage at which arrest occurs. Before that stage, ga1-3 stamen and anther development proceeds in a way that is indistinguishable from that of wild type. After that stage, wild-type development continues, while ga1-3 development is blocked. It will be interesting to determine if other GA-deficiency phenotypes (e.g. the particular shape of leaves of ga1-3 mutant plants) are also due to premature arrest of an identifiable developmental sequence.
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ACKNOWLEDGMENTS |
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Footnotes |
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