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Received 28 October 2008; revised 4 December 2008; accepted 8 December 2008; published online 21 January 2009.
*
For correspondence (fax +34 963877859; e-mail mpereza@ibmcp.upv.es).
†
Present address: Department of Plant Molecular Biology, University of Lausanne, Biophore Building, CH–1015 Lausanne, Switzerland.
Summary
Fruit development is usually triggered by ovule fertilization, and it requires coordination between seed
development and the growth and differentiation of the ovary to host the seeds. Hormones are known to
synchronize these two processes, but the role of each hormone, and the mechanism by which they interact, are
still unknown. Here we show that auxin and gibberellins (GAs) act in a hierarchical scheme. The synthetic
reporter construct DR5:GFP showed that fertilization triggered an increase in auxin response in the ovules,
which could be mimicked by blocking polar auxin transport. As the application of GAs did not affect auxin
response, the most likely sequence of events after fertilization involves auxin-mediated activation of GA
synthesis. We have confirmed this, and have shown that GA biosynthesis upon fertilization is localized
specifically in the fertilized ovules. Furthermore, auxin treatment caused changes in the expression of GA
biosynthetic genes similar to those triggered by fertilization, and also restricted to the ovules. Finally, GA
signaling was activated in ovules and valves, as shown by the rapid downregulation of the fusion protein RGA-
GFP after pollination and auxin treatment. Taken together, this evidence suggests a model in which
fertilization would trigger an auxin-mediated promotion of GA synthesis specifically in the ovule. The GAs
synthesized in the ovules would be then transported to the valves to promote GA signaling and thus
coordinate growth of the silique.
Introduction
Successful plant reproduction depends entirely on fruit-set, parthenocarpy are basic tools for the analysis of fruit
an essential process that can be defined as the activation of a development, as they facilitate the unraveling of the
developmental program which will convert the pistil into a molecular and genetic bases underlying fruit-set and early
developing fruit. This transition comprises two different and fruit development.
coordinated processes, namely the fertilization of the ovule Hormones seem to have a prominent role in synchroniz-
and the growth of the surrounding pistil and/or other ing fertilization and fruit growth. As early as the 1930s it was
structures to allocate the developing seeds (Gillaspy et al., proposed that fruit development would be initiated as a
1993). In most species, coordination between these two consequence of hormones synthesized in developing seeds
events is achieved because the signal that promotes fruit (Gustafson, 1936, 1939; Nitsch, 1950, 1952). Later work
growth originates only in developing seeds. However, the showed that early abortion of the fertilized ovules prevented
existence of fruits without seeds (parthenocarpic fruits) fruit development in pea, but fruit growth was restored by
indicates that the fertilization of the ovules is not an absolute application of several plant growth regulators (Eeuwens and
requirement, i.e. fruit development can be uncoupled from Schwabe, 1975). The involvement of hormones in fruit-set is
fertilization and seed development. Natural and induced thus supported by the major observation that exogenous
applications are sufficient to trigger fruit development, but tomato possess ARF8- and IAA9-related proteins that phys-
also by the fact that parthenocarpic mutants are generally ically interact to regulate fruit-set. Thus an ARF/IAA complex
affected in hormone biosynthesis and/or signaling. Gibber- would be directly involved in fruit initiation (Swain and
ellins (GAs), auxins and, in some cases, cytokinins have Koltunow, 2006; Pandolfini et al., 2007). Further evidence
been shown to be particularly efficient in several species to comes from the parthenocarpy displayed by transgenic
trigger fruit growth. Treatment of unpollinated pea ovaries plants with increased biosynthesis of the auxin indole-3-
with auxin [2,4-dichlorophenoxyacetic acid (2,4-D) and acetic acid (IAA) (Rotino et al., 1997; Yin et al., 2006;
4-chloroindole-3-acetic acid (4-Cl-IAA)], GA (GA1 or GA3), Costantini et al., 2007). Moreover, the tryptophan amino-
or cytokinin [6-benzylaminopurine (BAP)] results in the transferase TAA1, a key enzyme for auxin biosynthesis, is
development of parthenocarpic fruits (Garcia-Martinez and expressed in the apical parts of embryos, coinciding in
Carbonell, 1980; Ozga and Reinecke, 1999), although only location and time with the predicted sites of auxin produc-
GA3 treatment results in fruits that are nearly identical to tion upon fertilization (Stepanova et al., 2008).
fruits generated by pollination (Carbonell and Garcia-Marti- The observation that both auxins and GAs are involved in
nez, 1985). Remarkably, GAs and auxins have a synergistic early events that lead from fertilization to fruit development
effect on fruit growth when applied simultaneously (Ozga raises the question of whether these hormones act in
and Reinecke, 1999). Unlike in pea, auxin is the hormone parallel, regulating different aspects of the process, or in a
with a larger capacity to induce fruit-set in tomato, followed sequential manner, and what is the hierarchy and mecha-
by GAs (Homan, 1964; Alabadi et al., 1996). Pistils of nism of their interaction. Previous work suggests that GAs
Arabidopsis also respond to exogenous GAs, auxins and may act downstream of auxins. For instance, auxins have
cytokinins, although the treatments only induce partial fruit been shown to regulate the expression of several GA
development (Vivian-Smith and Koltunow, 1999). biosynthesis genes (Ross et al., 2000; Wolbang and Ross,
Endogenous bioactive GAs have recently been shown to 2001; O’Neill and Ross, 2002; Frigerio et al., 2006), and auxin
play a fundamental role in fruit development in Arabidopsis: is needed for GA signaling during root growth and apical
the GA biosynthetic enzymes GA 20-oxidases and GA 3- hook formation in the hypocotyl (Achard et al., 2003; Fu and
oxidases are required for silique elongation (Hu et al., 2008; Harberd, 2003). Furthermore, GAs are required for auxin-
Rieu et al., 2008b), and blocking GA inactivation, by knock- induced fruit set in tomato (Serrani et al., 2008). Neverthe-
ing out the five inactivating enzymes GA 2-oxidases, leads to less, the application of the two different hormones does not
the formation of parthenocarpic fruits in the absence of seem to be equally efficient in many systems, pointing to
fertilization (Rieu et al., 2008a). Moreover, tomato mutants specific roles for each hormone. Therefore, to uncover the
pat, pat2 and pat3/pat4 show enhanced expression of GA molecular mechanism by which GAs and auxins regulate
biosynthetic genes and increased levels of GAs, which in fruit-set in response to fertilization, and to determine the
turn induce parthenocarpic development (Fos et al., 2000, extent of crosstalk between these two hormones, we
2001; Olimpieri et al., 2007). In addition, GA concentration is decided to study the temporal and spatial regulation of the
much higher in the parthenocarpic citrus variety satsuma signaling events that occur during fruit-set in Arabidopsis.
than in the non-parthenocarpic self-incompatible clemen- We show that fertilization triggers an increase in auxin
tine, which again suggests that endogenous GA content is a response in ovules, which can be mimicked by blocking
limiting factor for parthenocarpic development (Talon et al., polar auxin transport. This induces subsequent activation of
1992). GA metabolism specifically in the young seeds. Further-
A role for auxin in promoting fruit-set and development more, GA signaling occurs in the valve, suggesting that GAs
has been revealed by recent work in Arabidopsis and synthesized in the seeds must be translocated to the pod to
tomato, species in which key elements in auxin signaling promote cell expansion and other differentiation processes.
with a function in fruit initiation and development have been Finally, we also describe the parthenocarpic phenotype of a
identified (Swain and Koltunow, 2006; Pandolfini et al., quadruple-DELLA loss-of-function mutant, which genetically
2007). In tomato, loss-of-function of the IAA9 gene, encoding confirms the role of GAs in fruit-set.
a nuclear-localized Aux/IAA protein, or of the auxin response
factor ARF7 result in plants with parthenocarpic fruits,
Results
suggesting that IAA9 and ARF7 inhibit growth in the absence
of fertilization (Wang et al., 2005; de Jong et al., 2008).
Hormonal regulation of fruit-set in Arabidopsis
Furthermore, in Arabidopsis, a loss-of-function allele of
ARF8 (fwf or arf8-4) also provokes parthenocarpic fruit To dissect the specific role of the different regulators of fruit-
development (Vivian-Smith et al., 2001; Goetz et al., 2006, set in autopollinated and self-compatible species it is nec-
2007). Proteins of the Aux/IAA and ARF families interact to essary to avoid fertilization. This is usually achieved by
mediate auxin signaling (Dharmasiri and Estelle, 2004), emasculating the flowers, but to carry out large-scale anal-
which leads to the hypothesis that both Arabidopsis and yses a more time-efficient procedure is needed. Therefore,
we chose the conditional male-sterile cer6-2 mutant of Ara- with the synthetic auxin 2,4-D also promoted fruit elonga-
bidopsis for our studies (Preuss et al., 1993; Fiebig et al., tion, although a slightly shorter fruit was obtained when
2000). At low relative humidity, mutant pollen grains do not compared with NAA or GA3 treatments (Figure 1). As
hydrate and fertilization does not take place. In our low- previously reported (Vivian-Smith and Koltunow, 1999),
humidity growth conditions, no seed set was observed in the the final fruit length was approximately 30–60% of that of a
cer6-2 mutant (data not shown). Mutant plants do not show pollinated pistil, although longer fruits were obtained when
any other apparent phenotype, remaining morphologically NAA and 2,4-D were applied 1 or 2 days after anthesis (data
identical to the Ler parental plant. not shown).
More importantly, the response of cer6-2 pistils to N-1-naphthylphthalamic acid (NPA), an inhibitor of polar
hormonal treatments was equivalent to that reported for auxin transport (Keitt and Baker, 1966; Geldner et al., 2001),
emasculated flowers in the Ler and Col-0 backgrounds provokes alteration of auxin levels in plant tissues (Ljung
(Vivian-Smith and Koltunow, 1999). Exogenous application et al., 2001; Desgagne-Penix et al., 2005), and has been used
of GA3 or naphthylacetic acid (NAA) to cer6-2 flowers under to test the interaction of auxin with other hormones (Fu and
low humidity at anthesis promoted parthenocarpic fruit Harberd, 2003). To test whether disruption of polar auxin
development, measured as the increase in fruit length transport had any effect on fruit-set, we applied NPA to cer6-
7 days after the treatment (Figure 1). Dose–response exper- 2 flowers at anthesis. A significant increase in fruit size was
iments showed these concentrations to be optimal for observed 7 days after the application, reaching a final length
inducing fruit-set (data not shown). In addition, treatment similar to that obtained by GA3 or NAA treatments (Fig-
ure 1). This indicates that a block in polar auxin transport is
able to induce the development of parthenocarpic fruits,
(a)
mimicking the effect of auxin application. Interestingly,
neither the single treatments nor combined treatments with
auxin (2,4-D or NAA) and GA yielded parthenocarpic siliques
of a size comparable with that of fertilized fruits (data not
shown). This suggests that factors other than hormones
might be necessary for full development of the fruit.
Alternatively, continuous hormone synthesis in the devel-
oping seeds throughout fruit development, as opposed to a
punctual treatment, might be required for fruits to attain
their final size.
These results confirm that GAs and auxins promote fruit
development in Arabidopsis, but do not clarify whether the
two hormones regulate different aspects of fruit-set or act
upon the same processes. To start dissecting this question,
(b) we examined the effect of the different treatments on the
tissue structure of the pistil. As shown in Figure 2, trans-
verse cryosections of the mock-treated pistils displayed the
usual layered structure (Ferrandiz et al., 1999; Roeder and
Yanofsky, 2006), composed of an external epidermis or
exocarp, followed by three layers of chlorenchyma cells or
mesocarp, and two layers of endocarp [the inner epidermis
or endocarp a (ena), and the endocarp b (enb)] which
becomes the lignified valve layer in mature fruits. This
overall structure was maintained in the fruits obtained by
hormonal application, with the exception of the ena cell
layer which was absent in the GA-induced fruits. The
Figure 1. Fruit-set is induced by blocking polar auxin transport with NPA. collapse of ena is known to occur in pollination-induced
cer6-2 flowers at anthesis were treated with mock, gibberellin (GA3), 2,4- fruit development, towards the end of developmental stage
dichlorophenoxyacetic acid (2,4-D), N-1-naphthylphthalamic acid (NPA), 17 (Roeder and Yanofsky, 2006); therefore GA3 treatment
naphthylacetic acid (NAA), or were hand-pollinated with Landsberg erecta
(Ler) pollen (Pol.). Pistils and fruits were harvested 7 days after the treatment. seems to accelerate the destruction of the ena layer. We
(a) Images of representative pistils and fruits. have ruled out the possibility that the premature collapse of
(b) Quantification of pistil and fruit length. Mean and SD were calculated from the ena is due to a specific effect of GA3 on the cer6-2
at least 25 pistils/fruits per treatment. The length of pistils and fruits was
normalized to the length of mock-treated pistils. The experiment was repeated mutation, given that the same collapse was observed
three times with similar results. in emasculated flowers of wild-type Ler plants treated with
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
Auxin–gibberellin regulation of fruit-set in Arabidopsis 321
Figure 2. Hormone-induced fruits show similar valve structure to those induced by pollination.
Transverse cryosections of pistils and fruits from cer6-2 (first and second rows) or Landsberg erecta (Ler) (third row) at 7 days post-anthesis. Pistils were treated with
mock, mock, gibberellin (GA3), 2,4-dichlorophenoxyacetic acid (2,4-D), N-1-naphthylphthalamic acid (NPA), naphthylacetic acid (NAA) or were hand-pollinated with
Ler pollen (Pol.). Flowers from Ler plants were emasculated 1 day before anthesis and treated on the day of anthesis. ex, exocarp (yellow); me, mesocarp (green);
enb, endocarp b (red); ena, endocarp a (blue). Absence of ena in GA3-treated pistils is indicated by an asterisk. Bars are 50 lm.
GA3 (Figure 2). These results suggest that at least one aspect which alleviates the repression they impose. To confirm that
of fruit development is under specific regulation by GAs, and GA-regulated DELLA proteins are involved in the control of
not auxins. fruit-set, we examined the pistil phenotype of a multiple
DELLA knockout mutant. As shown in Figure 3(a), while the
pistil length at anthesis of the quadruple gai-t6 rga-t2 rgl1-1
Constitutive GA signaling in a multiple DELLA
rgl2-1 mutant (Achard et al., 2006) was similar to the size of
loss-of-function mutant triggers fruit-set
pistils in wild-type plants, mutant pistils grew longer after
The five members of the DELLA gene family in Arabidopsis anthesis, reaching a final length similar to parthenocarpic
are repressors of GA signaling (Fleet and Sun, 2005). In the fruits obtained by GA3 treatment. In addition, constitutive
presence of GAs, DELLA proteins are rapidly degraded, GA signaling in the mutant plants also confers impaired
(a) (b)
12 Ler
Pistil or fruit length (mm)
12
Pistil or fruit length (mm)
Quadruple-DELLA
10
R 2 0.945
10
8
R 2 0.803
8
6
4 6
4 Ler
2
Quadruple-DELLA
0 2
Anthesis Unfertilized Fertilized 0 10 20 30 40 50 60 70
Seed number per fruit
Figure 5. Auxin response is detected in ovules upon fertilization or by treatments with auxin or N-1-naphthylphthalamic acid (NPA), but not with gibberellin (GA3).
Expression of GFP under the control of the DR5rev promoter in ovules and valves of pistils and fruits of cer6-2 plants. Flowers were emasculated and treated with
mock, GA3, 2,4-dichlorophenoxyacetic acid (2,4-D), or NPA, or hand-pollinated with Landsberg erecta (Ler) pollen (Pollinated). Images were taken 24 h later and are
either the composite of fluorescent GFP and bright field images (first row), or the fluorescent GFP image (second row). Chlorophyll fluorescence is observed as red
signal, while GFP appears as green signal. f, funiculus, ch, chalaza; i, integuments; m, micropyle.
(a)
12 AtGA20ox1
10
6
(b) AtGA3ox1
4
20
2
1 15
0
AtGA20ox2 10
8
5
6
0
4
6 AtGA3ox2
2 5
Relative gene expression level
60 1
0
40 AtGA3ox3
2000
20
1500
0
20 AtGA20ox4 1000
15 500
10 0
25
AtGA3ox4
5
20
0 15
AtGA20ox5
100 10
80 5
60 0
P P V O P VO P VO P V O PV O P V O
40 0h 24 h 48 h 72 h 24 h 48 h 72 h
20 Unfertilized Fertilized
0
P P V O P VO P VO P V O PV O P V O
0h 24 h 48 h 42 h 24 h 48 h 72 h
Unfertilized Fertilized
Figure 6. Expression of gibberellin (GA) biosynthesis genes, GA 20-oxidases and GA 3-oxidases, is upregulated in the ovule upon fruit-set.
Unfertilized (white bars) and fertilized (grey bars) cer6-2 pistils were harvested at 0, 24, 48, and 72 h after anthesis. Expression was analyzed in whole pistils (P), as
well as in manually dissected valves (V) and ovules (O) at each time point with the exception of the 0 h control sample.
(a) Expression of the five GA 20-oxidase genes.
(b) Expression of the four GA 3-oxidase genes. Each sample was a pool of at least 50 pistil/fruits. Quantitative RT-PCR data were normalized to the expression of
ACT8 (At1g49240) and then to the expression of control unpollinated whole pistils at anthesis. Expression of AtGA3ox3 at 0 h was not detected and a minimum Ct of
34 was assigned at this time point for quantification purposes. Data correspond to mean and SD of three technical replicates. Two independent experiments were
carried out, with similar results.
Elevated GA levels trigger GA signaling by promoting regulators upon GA responses (Fleet and Sun, 2005). In a
the degradation of DELLA proteins in a cell-autonomous reciprocal manner, the examination of the stability of DELLA
manner, relieving the restriction imposed by these negative proteins is a suitable approach to infer changes in GA
AtGA2ox1
8
2 AtGA2ox6
6
1
0
AtGA2ox2 4
80
2
60
1
40 0
20
AtGA2ox7
10
0 8
AtGA2ox3
10 6
8 4
6 2
4 0
5
AtGA2ox8
2
4
0
AtGA2ox4 3
4
2
3
1
2
0
P P V O P V O P V O P V O P V O P V O
1 0h 24 h 48 h 72 h 24 h 48 h 72 h
Unfertilized Fertilized
0
P P V O P V O P V O P V O P V O P V O
0h 24 h 48 h 72 h 24 h 48 h 72 h
Unfertilized Fertilized
Figure 7. Expression of gibberellin (GA) inactivation genes, GA 2-oxidases, is upregulated in the ovule and valve upon fruit-set.
Samples were those used in Figure 6, and the experimental procedures are described in the figure legend.
concentration, although there are publications that suggest was the most effective, so a low signal was detected 24 h
that signals other than GA might affect DELLA levels. after the treatment, while in 2,4-D-treated and pollinated
Therefore, we examined the level of the fusion protein pistils the signal decreased after 48 h. In the case of
GFP-RGA, expressed under the control of the endogenous pollination, a clear correlation was found between the
RGA promoter (ProRGA:GFP-RGA) (Silverstone et al., presence/absence of GFP-RGA and non-fertilized/fertilized
2001), in pistils in response to various treatments that ovules (Figure 9). In all cases, GFP-RGA was also degraded
induce fruit-set. At anthesis, or 1 day after mock treatment, in the valve, with a time course similar to the disappear-
GFP-RGA was localized in ovules, with the highest signal at ance in the ovule.
the base of the ovule and funiculus (Figure 9). It could also Our data suggest that RGA degradation in fertilized ovules
be detected in the valves, although at a lower intensity. As during fruit-set is most probably due to elevated GA levels,
expected, exogenous GA3 provoked the complete disap- achieved by auxin-induced upregulation of GA biosynthesis
pearance of GFP signal in ovules and valves 24 h after the genes after fertilization. Interestingly, RGA degradation is
treatment. More importantly, pollination and NPA and 2,4- ovule/seed autonomous (Figure 9), suggesting that no GA
D treatments also caused degradation of RGA in seeds/ diffusion occurs from fertilized to unfertilized ovules. In
ovules and in valves, albeit with different kinetics. NPA addition, degradation of RGA in the valves indicates the
(a) (b)
30
AtGA20ox1 AtGA20ox1
6
20
4
10
2
0 0
4
AtGA20ox2 16 AtGA20ox2
3
12
2
8
1 4
0 0
8
AtGA3ox1 AtGA3ox1
12
8
4
4
2
1
0 0
2 6 24 48 2 6 24 48 2 6 24 48 h P V O P V O P V O P V O
GA3 NPA 2,4-D Mock GA3 NPA 2,4-D
Figure 8. Expression of gibberellin (GA) biosynthesis genes is upregulated upon fruit-set induced by auxin in an organ-specific manner.
(a) Time course of induction. Gene expression is shown for AtGA20ox1, AtGA20ox2, and AtGA3ox1 in fruits of cer6-2 at 2, 6, 24, and 48 h after treatment with mock,
GA3, N-1-naphthylphthalamic acid (NPA), or 2,4-dichlorophenoxyacetic acid (2,4-D).
(b) Tissue specificity of the expression. Gene expression is shown for AtGA20ox1, AtGA20ox2, and AtGA3ox1 in cer6-2 whole pistils (P), valves (V), and ovules (O) at
6 h after treatment with mock, GA3, NPA, or 2,4-D. Each sample was a pool of at least 50 pistils/fruits.
Quantitative RT-PCR data in (a) were normalized to the expression of PP2A (At1g13320) and then to the expression of mock-treated pistils at the same time-point.
Data in (b) were normalized to the expression of ACT8 and then to the expression of mock-treated whole pistils. Data correspond to mean and SD of three technical
replicates. Two independent experiments were carried out, with similar results.
presence of GAs in that tissue. Since our expression analysis olism specifically in fertilized ovules; (iii) GA signaling is
(Figure 6) does not support upregulation of GA synthesis in detected in both seeds and valves; and (iv) constitutive GA
the valves, a likely possibility is that they are translocated signaling is sufficient to promote parthenocarpic fruit
from the fertilized ovules, where they are synthesized development.
according to our gene expression data.
Auxin response in the fertilized ovule is an early
Discussion event during fruit-set
Our analysis of the molecular events associated with the Through the analysis of ProDR5rev:GFP transgenic plants,
action of auxin and GA during fruit-set indicates that: we have shown that an auxin response is activated in ovules
(i) auxin response in young seeds is an early event during soon after fertilization, probably as a consequence of
fruit-set; (ii) auxin promotes the activation of GA metab- elevated auxin levels. Several explanations could account
for this observation: a local increase in auxin synthesis, Finally, overexpressing IAA-biosynthesis genes in ovules or
a blockage of auxin transport, interference with auxin placenta of transgenic plants result in parthenocarpy in
response upon fertilization, or even the downloading of several species (for example, Rotino et al., 1997; Yin et al.,
auxins from the pollen tube into the ovule (Sweet and Lewis, 2006). In summary, either application of auxin or overex-
1969). However, the fact that NPA treatment induces par- pression of auxin biosynthetic genes in ovules would
thenocarpic fruit development by mimicking the same auxin promote the auxin response observed upon fertilization. It
response in the ovule seen upon fertilization rules out the is then plausible to hypothesize that elevated levels of auxin
latter two possibilities. Parthenocarpic development in the ovule would initiate a cascade of events that would
induced by a block in polar auxin transport was previously finally promote fruit growth. These events would include
reported in cucumber (Robinson et al., 1971; Beyer and auxin signaling, through the function of Aux/IAA and ARF
Quebedeaux, 1974), and it was proposed that the effect proteins (Swain and Koltunow, 2006; Pandolfini et al.,
might be due to a blockade in the outward flow of auxin from 2007), and crosstalk with other hormones (Weiss and Ori,
the ovary, resulting in auxin accumulation sufficient to 2007).
trigger fruit-set in absence of fertilization.
Whether the increase in auxin in the fertilized ovule is
Auxin action is mediated by gibberellins in the ovary
due to enhanced biosynthesis or interference in transport
cannot be determined based on our results. Nevertheless, Although the efficiency of the different hormones in the
we have not observed any modification in the abundance induction of fruit development varies between species, the
and localization of several PIN auxin efflux carriers (Blakes- activation of GA metabolism after fertilization is a common
lee et al., 2005) during pistil development and fertilization theme. For instance, early fruit development in tomato
(data not shown). Thus, it seems likely that auxin synthesis plants is characterized by elevated mRNA levels of copalyl
is activated in the ovule early after fertilization and that this diphosphate synthase, a gene involved in the early steps of
may initiate fruit development. Several pieces of evidence GA biosynthesis, LeGA20ox1 and LeGA20ox3, accompanied
support this view. First, auxins are naturally present in by decreased level of LeGA3ox2 (Rebers et al., 1999). Indeed,
developing ovules. We have detected a very low GFP signal the expression of GA 20-oxidase genes seems to be a lim-
driven by the ProDR5 in mock-treated ovules, which indi- iting step for the increase in GA concentration during fruit-
cates that a basal level of auxin is present in unfertilized set (Serrani et al., 2007). A similar trend has been observed
ovules. In addition, conjugated auxins have been detected in pea, where a GA 20-oxidase gene is strongly upregulated
in the ovules of unfertilized pistils (Aloni et al., 2006) and in during early fruit development, both in the developing seeds
developing ovules and embryos (Benkova et al., 2003; Friml and in the pod (Garcia-Martinez et al., 1997).
et al., 2003). Second, exogenous auxin can induce parthe- How does fertilization activate GA biosynthesis? Our
nocarpy in a variety of species, including Arabidopsis. results suggest that the initial event that causes this
GA20ox4
GA20ox5 accelerated in GA-induced fruits. Similar data were reported
in emasculated spy-4 pistils induced to grow with GA3, but
GA3ox1
not in wild-type pistils (Vivian-Smith and Koltunow, 1999).
GA3ox2
GA3ox3 These data suggest that degradation of ena prior to pod
GA3ox4 shattering is controlled by GAs, as is the case for
other developmental processes that take place during fruit
GA2ox1
development, such as cell expansion in mesocarp cells
GA2ox2
GA2ox3 (Vivian-Smith and Koltunow, 1999).
GA2ox4
GA2ox6
GA2ox7 Experimental procedures
GA2ox8
Ovule Plant material and hormone treatments
Arabidopsis thaliana cer6-2 seeds, in the Landsberg erecta (Ler)
GA20ox1 accession (Preuss et al., 1993; Fiebig et al., 2000), were obtained
GA20ox2
Valve from the Arabidopsis Biological Resource Center (ABRC). Seeds
GA20ox3 from the lines ProDR5rev:GFP in the Columbia-0 accession (Col-0)
GA20ox4 (Benkova et al., 2003), the ProRGA:GFP-RGA in Ler (Silverstone
GA20ox5 et al., 2001), and the quadruple-DELLA mutant (gai-t6 rga-t2 rgl1-1
rgl2-1) in Ler (Achard et al., 2006) were obtained from Jiri Friml
GA3ox1 (Tübingen University, Germany), Tai-ping Sun (Duke University,
GA3ox2 NC, USA), and Nicholas P. Harberd (John Innes Centre, Norwich,
GA3ox3 UK), respectively.
GA3ox4 Plants were grown in chambers at 22C under a 16-h light/8-h dark
photoperiod and at 50% relative humidity to avoid fertilization of
GA2ox1 cer6-2. For each plant, only flowers from the primary bolt, between 0
GA2ox2 and 2 days post-anthesis (dpa) were used. For plants not harboring
GA2ox3 the mutant cer6-2 allele (i.e. the parental Ler, the transgenic line
GA2ox4 harboring the ProDR5rev:GFP construct, and the quadruple-DELLA
GA2ox6
mutant), self-pollination was avoided by emasculation 1 day before
GA2ox7
anthesis. Fruit-set was induced by hand pollination with Ler pollen
GA2ox8
or spray application of 330 lM GA3 (Fluka, http://www.sigmaaldrich.
com/), 10 lM 2,4-D (Sigma Aldrich, http://www.sigmaaldrich.
com/), 300 lM NAA (Sigma), or 50 lM NPA (Duchefa Biochemie,
Figure 10. Schematic representation of the temporal and spatial expression http://www.duchefa.com/). These concentrations were tested to be
of gibberellin (GA) metabolism genes upon fruit-set.
optimal to induce fruit-set. Tween-80 at 0.05% was used as the
Arrows indicate the temporal expression frame of the corresponding gene,
wetting agent, and the pH was adjusted between 6.5 and 7.0.
between anthesis and 72 h after fertilization, in the ovule (upper panel) and
valve (lower panel). Samples were harvested and processed at the indicated time-
points. For dissection experiments, valves and ovules were col-
lected by hand dissection under a stereoscope microscope with the
help of a razorblade and acupuncture needles, placed in a vial with
parthenocarpic siliques (Rieu et al., 2008a) implies that RNA extraction buffer, and RNA was immediately extracted (see
even in the absence of fertilization the pistil must have below).
access to GAs that would then accumulate and drive To test induction of fruit-set, pistil or fruit length was measured at
parthenocarpic fruit development. Our results suggest that 7 dpa or at full maturity. Pistils and fruits were harvested and
scanned, and images were analyzed using Image J software
these active GAs would have to be transported to the
(Abramoff et al., 2004).
silique from other tissues, but specific experiments would
be needed to address this question.
Finally, our data show that while GA synthesis occurs only Scanning electron microscopy (SEM)
in the young seed (Figure 10), GA signaling is detected both Samples were harvested, mounted on the specimen holder of a CT-
in developing seeds and in valves, indicating that GAs could 1000C cryo-transfer system (Oxford Instruments, http://www.
be the hypothesized growth-regulator that coordinates seed oxford-instruments.com/), interfaced with a JEOL JSM-5410
and fruit development. The role of GAs in the valves would scanning electron microscope, and frozen in liquid N2. Samples
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