The Plant Journal (2009) 58, 318–332
doi: 10.1111/j.1365-313X.2008.03781.x
Fertilization-dependent auxin response in ovules triggers
fruit development through the modulation of gibberellin
metabolism in Arabidopsis
Eavan Dorcey†, Cristina Urbez, Miguel A. Blázquez, Juan Carbonell and Miguel A. Perez-Amador*
Instituto de Biologı́a Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cientı́ficas,
Universidad Politécnica de Valencia (CSIC-UPV), Avenida de los naranjos s/n, 46022 Valencia, Spain
Received 28 October 2008; revised 4 December 2008; accepted 8 December 2008; published online 21 January 2009.
*
For correspondence (fax +34 963877859; e-mail [email protected]).
†
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 RGAGFP 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.
Keywords: Fruit-set, auxin, gibberellin, crosstalk, Arabidopsis.
Introduction
Successful plant reproduction depends entirely on fruit-set,
an essential process that can be defined as the activation of a
developmental program which will convert the pistil into a
developing fruit. This transition comprises two different and
coordinated processes, namely the fertilization of the ovule
and the growth of the surrounding pistil and/or other
structures to allocate the developing seeds (Gillaspy et al.,
1993). In most species, coordination between these two
events is achieved because the signal that promotes fruit
growth originates only in developing seeds. However, the
existence of fruits without seeds (parthenocarpic fruits)
indicates that the fertilization of the ovules is not an absolute
requirement, i.e. fruit development can be uncoupled from
fertilization and seed development. Natural and induced
318
parthenocarpy are basic tools for the analysis of fruit
development, as they facilitate the unraveling of the
molecular and genetic bases underlying fruit-set and early
fruit development.
Hormones seem to have a prominent role in synchronizing fertilization and fruit growth. As early as the 1930s it was
proposed that fruit development would be initiated as a
consequence of hormones synthesized in developing seeds
(Gustafson, 1936, 1939; Nitsch, 1950, 1952). Later work
showed that early abortion of the fertilized ovules prevented
fruit development in pea, but fruit growth was restored by
application of several plant growth regulators (Eeuwens and
Schwabe, 1975). The involvement of hormones in fruit-set is
thus supported by the major observation that exogenous
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd
Auxin–gibberellin regulation of fruit-set in Arabidopsis 319
applications are sufficient to trigger fruit development, but
also by the fact that parthenocarpic mutants are generally
affected in hormone biosynthesis and/or signaling. Gibberellins (GAs), auxins and, in some cases, cytokinins have
been shown to be particularly efficient in several species to
trigger fruit growth. Treatment of unpollinated pea ovaries
with auxin [2,4-dichlorophenoxyacetic acid (2,4-D) and
4-chloroindole-3-acetic acid (4-Cl-IAA)], GA (GA1 or GA3),
or cytokinin [6-benzylaminopurine (BAP)] results in the
development of parthenocarpic fruits (Garcia-Martinez and
Carbonell, 1980; Ozga and Reinecke, 1999), although only
GA3 treatment results in fruits that are nearly identical to
fruits generated by pollination (Carbonell and Garcia-Martinez, 1985). Remarkably, GAs and auxins have a synergistic
effect on fruit growth when applied simultaneously (Ozga
and Reinecke, 1999). Unlike in pea, auxin is the hormone
with a larger capacity to induce fruit-set in tomato, followed
by GAs (Homan, 1964; Alabadi et al., 1996). Pistils of
Arabidopsis also respond to exogenous GAs, auxins and
cytokinins, although the treatments only induce partial fruit
development (Vivian-Smith and Koltunow, 1999).
Endogenous bioactive GAs have recently been shown to
play a fundamental role in fruit development in Arabidopsis:
the GA biosynthetic enzymes GA 20-oxidases and GA 3oxidases are required for silique elongation (Hu et al., 2008;
Rieu et al., 2008b), and blocking GA inactivation, by knocking out the five inactivating enzymes GA 2-oxidases, leads to
the formation of parthenocarpic fruits in the absence of
fertilization (Rieu et al., 2008a). Moreover, tomato mutants
pat, pat2 and pat3/pat4 show enhanced expression of GA
biosynthetic genes and increased levels of GAs, which in
turn induce parthenocarpic development (Fos et al., 2000,
2001; Olimpieri et al., 2007). In addition, GA concentration is
much higher in the parthenocarpic citrus variety satsuma
than in the non-parthenocarpic self-incompatible clementine, which again suggests that endogenous GA content is a
limiting factor for parthenocarpic development (Talon et al.,
1992).
A role for auxin in promoting fruit-set and development
has been revealed by recent work in Arabidopsis and
tomato, species in which key elements in auxin signaling
with a function in fruit initiation and development have been
identified (Swain and Koltunow, 2006; Pandolfini et al.,
2007). In tomato, loss-of-function of the IAA9 gene, encoding
a nuclear-localized Aux/IAA protein, or of the auxin response
factor ARF7 result in plants with parthenocarpic fruits,
suggesting that IAA9 and ARF7 inhibit growth in the absence
of fertilization (Wang et al., 2005; de Jong et al., 2008).
Furthermore, in Arabidopsis, a loss-of-function allele of
ARF8 (fwf or arf8-4) also provokes parthenocarpic fruit
development (Vivian-Smith et al., 2001; Goetz et al., 2006,
2007). Proteins of the Aux/IAA and ARF families interact to
mediate auxin signaling (Dharmasiri and Estelle, 2004),
which leads to the hypothesis that both Arabidopsis and
tomato possess ARF8- and IAA9-related proteins that physically interact to regulate fruit-set. Thus an ARF/IAA complex
would be directly involved in fruit initiation (Swain and
Koltunow, 2006; Pandolfini et al., 2007). Further evidence
comes from the parthenocarpy displayed by transgenic
plants with increased biosynthesis of the auxin indole-3acetic acid (IAA) (Rotino et al., 1997; Yin et al., 2006;
Costantini et al., 2007). Moreover, the tryptophan aminotransferase TAA1, a key enzyme for auxin biosynthesis, is
expressed in the apical parts of embryos, coinciding in
location and time with the predicted sites of auxin production upon fertilization (Stepanova et al., 2008).
The observation that both auxins and GAs are involved in
early events that lead from fertilization to fruit development
raises the question of whether these hormones act in
parallel, regulating different aspects of the process, or in a
sequential manner, and what is the hierarchy and mechanism of their interaction. Previous work suggests that GAs
may act downstream of auxins. For instance, auxins have
been shown to regulate the expression of several GA
biosynthesis genes (Ross et al., 2000; Wolbang and Ross,
2001; O’Neill and Ross, 2002; Frigerio et al., 2006), and auxin
is needed for GA signaling during root growth and apical
hook formation in the hypocotyl (Achard et al., 2003; Fu and
Harberd, 2003). Furthermore, GAs are required for auxininduced fruit set in tomato (Serrani et al., 2008). Nevertheless, the application of the two different hormones does not
seem to be equally efficient in many systems, pointing to
specific roles for each hormone. Therefore, to uncover the
molecular mechanism by which GAs and auxins regulate
fruit-set in response to fertilization, and to determine the
extent of crosstalk between these two hormones, we
decided to study the temporal and spatial regulation of the
signaling events that occur during fruit-set in Arabidopsis.
We show that fertilization triggers an increase in auxin
response in ovules, which can be mimicked by blocking
polar auxin transport. This induces subsequent activation of
GA metabolism specifically in the young seeds. Furthermore, GA signaling occurs in the valve, suggesting that GAs
synthesized in the seeds must be translocated to the pod to
promote cell expansion and other differentiation processes.
Finally, we also describe the parthenocarpic phenotype of a
quadruple-DELLA loss-of-function mutant, which genetically
confirms the role of GAs in fruit-set.
Results
Hormonal regulation of fruit-set in Arabidopsis
To dissect the specific role of the different regulators of fruitset in autopollinated and self-compatible species it is necessary to avoid fertilization. This is usually achieved by
emasculating the flowers, but to carry out large-scale analyses a more time-efficient procedure is needed. Therefore,
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
320 Eavan Dorcey et al.
we chose the conditional male-sterile cer6-2 mutant of Arabidopsis for our studies (Preuss et al., 1993; Fiebig et al.,
2000). At low relative humidity, mutant pollen grains do not
hydrate and fertilization does not take place. In our lowhumidity growth conditions, no seed set was observed in the
cer6-2 mutant (data not shown). Mutant plants do not show
any other apparent phenotype, remaining morphologically
identical to the Ler parental plant.
More importantly, the response of cer6-2 pistils to
hormonal treatments was equivalent to that reported for
emasculated flowers in the Ler and Col-0 backgrounds
(Vivian-Smith and Koltunow, 1999). Exogenous application
of GA3 or naphthylacetic acid (NAA) to cer6-2 flowers under
low humidity at anthesis promoted parthenocarpic fruit
development, measured as the increase in fruit length
7 days after the treatment (Figure 1). Dose–response experiments showed these concentrations to be optimal for
inducing fruit-set (data not shown). In addition, treatment
(a)
(b)
Figure 1. Fruit-set is induced by blocking polar auxin transport with NPA.
cer6-2 flowers at anthesis were treated with mock, gibberellin (GA3), 2,4dichlorophenoxyacetic acid (2,4-D), N-1-naphthylphthalamic acid (NPA),
naphthylacetic acid (NAA), or were hand-pollinated with Landsberg erecta
(Ler) pollen (Pol.). Pistils and fruits were harvested 7 days after the treatment.
(a) Images of representative pistils and fruits.
(b) Quantification of pistil and fruit length. Mean and SD were calculated from
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
three times with similar results.
with the synthetic auxin 2,4-D also promoted fruit elongation, although a slightly shorter fruit was obtained when
compared with NAA or GA3 treatments (Figure 1). As
previously reported (Vivian-Smith and Koltunow, 1999),
the final fruit length was approximately 30–60% of that of a
pollinated pistil, although longer fruits were obtained when
NAA and 2,4-D were applied 1 or 2 days after anthesis (data
not shown).
N-1-naphthylphthalamic acid (NPA), an inhibitor of polar
auxin transport (Keitt and Baker, 1966; Geldner et al., 2001),
provokes alteration of auxin levels in plant tissues (Ljung
et al., 2001; Desgagne-Penix et al., 2005), and has been used
to test the interaction of auxin with other hormones (Fu and
Harberd, 2003). To test whether disruption of polar auxin
transport had any effect on fruit-set, we applied NPA to cer62 flowers at anthesis. A significant increase in fruit size was
observed 7 days after the application, reaching a final length
similar to that obtained by GA3 or NAA treatments (Figure 1). This indicates that a block in polar auxin transport is
able to induce the development of parthenocarpic fruits,
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 developing 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,
we examined the effect of the different treatments on the
tissue structure of the pistil. As shown in Figure 2, transverse 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
collapse of ena is known to occur in pollination-induced
fruit development, towards the end of developmental stage
17 (Roeder and Yanofsky, 2006); therefore GA3 treatment
seems to accelerate the destruction of the ena layer. We
have ruled out the possibility that the premature collapse of
the ena is due to a specific effect of GA3 on the cer6-2
mutation, given that the same collapse was observed
in emasculated flowers of wild-type Ler plants treated with
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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.
which alleviates the repression they impose. To confirm that
GA-regulated DELLA proteins are involved in the control of
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
rgl2-1 mutant (Achard et al., 2006) was similar to the size of
pistils in wild-type plants, mutant pistils grew longer after
anthesis, reaching a final length similar to parthenocarpic
fruits obtained by GA3 treatment. In addition, constitutive
GA signaling in the mutant plants also confers impaired
GA3 (Figure 2). These results suggest that at least one aspect
of fruit development is under specific regulation by GAs, and
not auxins.
Constitutive GA signaling in a multiple DELLA
loss-of-function mutant triggers fruit-set
The five members of the DELLA gene family in Arabidopsis
are repressors of GA signaling (Fleet and Sun, 2005). In the
presence of GAs, DELLA proteins are rapidly degraded,
(a)
(b)
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Pistil or fruit length (mm)
Pistil or fruit length (mm)
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Unfertilized
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Figure 3. Quadruple–DELLA mutant shows parthenocarpic fruit development.
(a) Quantification of pistil and fruit length. Flowers of quadruple-DELLA mutant (gai-t6 rga-t2 rgl1-1 rgl2-1) and parental Landsberg erecta (Ler) plants were
emasculated and either allowed to grow in the absence of pollination (unfertilized) or hand pollinated (fertilized) with Ler pollen. Pistil and fruit length were
measured at anthesis, at 7 days after anthesis for unpollinated pistils or at maturity (stage 18) for pollinated pistils. The mean and SD were calculated from at least 50
pistils or fruits per treatment.
(b) Correlation between seed number and length of pistil or fruit of the quadruple-DELLA and Ler. Flowers were emasculated and hand pollinated with different
amount of Ler pollen to vary seed set. More than 40 fruits and pistils were used for each genotype. The correlation coefficient (R2) and adjusted regression line is
indicated. The experiment was repeated twice with similar results.
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Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
322 Eavan Dorcey et al.
Activation of auxin response in ovules upon fertilization
Figure 4. Degradation of endocarp a (ena) is mediated by GAs.
Transverse cryosections of fruits and pistils from quadruple-DELLA mutant
and Landsberg erecta (Ler) plants at 7 days post-anthesis. Cell layers of the
pericarp are indicated as in Figure 2. Absence of ena in the parthenocarpic
fruit of quadruple-DELLA is indicated by an asterisk. Bars are 50 lm.
fertilization (seed set) and silique elongation, because fruits
obtained by manual pollination were smaller in the quadruple-DELLA mutant than in the wild type (Figure 3a) and
contained fewer seeds (Figure 3b).
Consistent with the GA3 treatments, the ena layer in pistils
of the quadruple-DELLA mutant, which is present at anthesis, was absent 7 days later, while it remained unaltered in
wild-type pistils (Figure 4). This observation strongly supports the hypothesis that the degradation of the ena cell
layer is mediated by GAs.
The observation that impairment of polar auxin transport
causes parthenocarpic fruit development (Figure 1) suggests that fertilization could cause a local increase in the
concentration of endogenous auxin sufficient to trigger
fruit development. To test this hypothesis, we examined
the pattern of expression of the synthetic auxin-regulated
promoter DR5rev (Ulmasov et al., 1997) fused to GFP
(ProDR5rev:GFP) (Benkova et al., 2003) during fruit-set, as
an indirect indicator of auxin level and/or response. The
low-fluorescence signal detected at anthesis (data not
shown), and 24 h after mock treatment (Figure 5) was
associated with the lignin in the vasculature of the funiculus, while only a very low GFP signal was detected in
ovules. No GFP signal was detected in the valves. More
interestingly, 24 h after pollination, a strong increase in the
GFP signal was detected in the funiculus, chalaza, and
micropyle of the ovule, but not in the valve, which indicates that an increase in auxin response in the young
seeds is an early event in fruit development. DR5rev was
also strongly upregulated 24 h after 2,4-D application, and
this expression extended into the valve, which seems to be
the result of ectopic presence of the synthetic auxin. The
upregulation in ovules mimics the effect of pollination,
suggesting that an increase in auxin response in the
ovules is concomitant with their fertilization. This is further
supported by the observation that NPA treatment also
caused an upregulation of DR5rev expression in ovules but
not in valves (Figure 5). In contrast, ovules of GA3-treated
pistils did not show any GFP signal, indicating that GAinduced fruit development is independent of auxin in the
ovule. Two possible scenarios emerge: either auxin and
GAs act through independent pathways to promote fruit
development, or GAs largely mediate the promotion of
fruit growth induced by auxin.
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.
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Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
Auxin–gibberellin regulation of fruit-set in Arabidopsis 323
Gibberellin metabolism is activated by auxin after
fertilization
The possibility that GAs mediate the action of auxins in
Arabidopsis fruit-set is supported by previous reports
showing that exogenous auxin increases the production of
GAs in pea fruit pericarp (Van Huizen et al., 1995, 1997; Ozga
and Reinecke, 1999; Ozga et al., 2003). In tomato, it has
recently been shown that GAs are required for auxin-induced fruit-set (Serrani et al., 2008). Furthermore, auxin
upregulates GA-metabolism genes in Arabidopsis seedlings
(Frigerio et al., 2006). These observations led us to hypothesize that, upon fruit-set, auxin accumulation in ovules
would alter GA metabolism and GA levels, which would in
turn lead to fruit growth.
Using quantitative real-time PCR (qRT-PCR) we analyzed
the expression of the genes involved in GA metabolism in
pistils. Out of the 16 genes for GA metabolism described in
Arabidopsis (five encoding GA 20-oxidases, four encoding
GA 3-oxidases, and seven encoding GA 2-oxidases) (Curaba
et al., 2004; Frigerio et al., 2006), we could detect expression
for 15 of them in pistils at the anthesis stage (Figure S1). The
highest expression level was that of AtGA20ox2, followed by
AtGA2ox2, AtGA2ox4, and AtGA2ox6. Other genes were
expressed at similar levels to that of AtGA20ox1 (AtGA3ox1,
AtGA2ox1, and AtGA2ox8), while the rest showed lower or
very low expression. Overall, these data are in agreement
with previous data for the expression of AtGA20ox1 and
AtGA20ox2 (Rieu et al., 2008b) and GA 3-oxidases (Mitchum
et al., 2006; Hu et al., 2008).
To investigate the spatial and temporal expression
patterns of the genes involved in GA metabolism and to
understand the contribution of the different tissues within
the pistil to the coordination of fruit growth, we have
determined their expression level by qRT-PCR in manually
dissected pistils and fruits. Pistils were either hand-pollinated with wild-type pollen, or not pollinated, harvested 1,
2, and 3 days after anthesis, and dissected to separate
valves and ovules/seeds. Expression of most of the GA
biosynthetic genes was upregulated upon fertilization
within different time frames (Figure 6); but most importantly, induction occurred exclusively in the fertilized
ovules, not in the valves. One day after pollination,
AtGA20ox1 and AtGA3ox1 were transiently upregulated,
while other genes (AtGA20ox3, AtGA20ox5, AtGA3ox3,
and AtGA3ox4) were upregulated later. AtGA20ox2
showed a slight transient increase 2 days after pollination.
In contrast, expression of GA inactivation genes was
upregulated in both valves (AtGA2ox1 and AtGA2ox8)
and in fertilized ovules (AtGA2ox1, AtGA2ox3, and AtGA2ox4) (Figure 7). AtGA2ox1 was upregulated 24 h after
pollination, and remained elevated for the next 2 days in
both valves and seeds, while AtGA2ox3 and AtGA2ox4
were upregulated only in the seeds 72 h after pollination,
and AtGA2ox8 was upregulated only in the valves during
the first day (Figure 7).
Given that the expression of GA metabolism genes
during fruit-set is under strict spatial control, the question
arises whether auxin in the fertilized ovules directs this
regulation. If this is the case, auxin application, and
treatments with NPA, should also upregulate GA-metabolism genes in pollinated pistils. Application of auxin to
unpollinated pistils indeed produced an overall induction
of GA biosynthesis genes (Figure 8a, and Figures S2–S4 in
Supporting Information). In a similar way, NPA treatment,
that had been shown to increase auxin response in ovules
(Figure 5), also resulted in the immediate upregulation of
GA-metabolism genes. These effects were particularly
evident for AtGA20ox1, AtGA20ox2, and AtGA3ox1. On
the other hand, GA treatments caused a strong inhibition
of the expression of GA biosynthesis genes as a result
of negative feedback. Moreover, expression analysis in
dissected pistils indicated that several of these genes
responded specifically in ovules versus valves (Figures 8b
and S5–7): while AtGA20ox1 expression was induced by
auxin treatments in both ovules and valves, AtGA20ox2,
AtGA20ox3, and AtGA3ox1, were induced in the ovules,
and AtGA20ox4 and AtGA20ox5 were not significantly
affected. Finally, the GA 2-oxidase genes showed little or
no induction by auxin.
All these data suggest that, upon fertilization, auxin in
the ovule causes a rapid increase in expression of GA
biosynthesis genes that, in turn, results in an increased
production of GAs specifically in the seed. Although auxin
treatment mimics the effect of fertilization, there are
some differences, such as the auxin-induced expression
of AtGA20ox1 in the valve. This coincides with the effect of
4-Cl-IAA treatment on PsGA20ox1 expression in pea pericarp (Ngo et al., 2002), and may be attributed to an ectopic
induction due to the direct application of auxin to the pistil
wall. Thus, auxins and GAs seem to act in a sequential
manner to induce fruit growth after fertilization. Nevertheless, the existence of a parallel pathway, whereby GA
biosynthesis would be activated directly by fertilization
without auxin mediation, could not be completely ruled out
based on our data.
Stability of RGA protein in the ovules is controlled by auxin
Gibberellins are thought to exert their action in the valves,
where they promote cell expansion (and eventually the
collapse of the ena layer). But the fertilization-dependent
increase in auxin response occurs in the ovules (Figure 5),
where it provokes changes in GA metabolism. One relevant
question is whether this change in GA biosynthesis is
accompanied by changes in GA signaling and, more
importantly, whether GA signaling occurs only in the ovules
or also in the valves.
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Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
324 Eavan Dorcey et al.
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PV O
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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
the degradation of DELLA proteins in a cell-autonomous
manner, relieving the restriction imposed by these negative
regulators upon GA responses (Fleet and Sun, 2005). In a
reciprocal manner, the examination of the stability of DELLA
proteins is a suitable approach to infer changes in GA
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 325
AtGA2ox1
8
6
4
AtGA2ox6
2
1
0
6
AtGA2ox2
4
Relative gene expression level
Relative gene expression level
80
60
40
20
0
AtGA2ox3
10
8
6
4
2
2
1
0
AtGA2ox7
10
8
6
4
2
0
5
AtGA2ox8
4
0
4
AtGA2ox4
3
2
3
1
2
0
1
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
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
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
that signals other than GA might affect DELLA levels.
Therefore, we examined the level of the fusion protein
GFP-RGA, expressed under the control of the endogenous
RGA promoter (ProRGA:GFP-RGA) (Silverstone et al.,
2001), in pistils in response to various treatments that
induce fruit-set. At anthesis, or 1 day after mock treatment,
GFP-RGA was localized in ovules, with the highest signal at
the base of the ovule and funiculus (Figure 9). It could also
be detected in the valves, although at a lower intensity. As
expected, exogenous GA3 provoked the complete disappearance of GFP signal in ovules and valves 24 h after the
treatment. More importantly, pollination and NPA and 2,4D treatments also caused degradation of RGA in seeds/
ovules and in valves, albeit with different kinetics. NPA
was the most effective, so a low signal was detected 24 h
after the treatment, while in 2,4-D-treated and pollinated
pistils the signal decreased after 48 h. In the case of
pollination, a clear correlation was found between the
presence/absence of GFP-RGA and non-fertilized/fertilized
ovules (Figure 9). In all cases, GFP-RGA was also degraded
in the valve, with a time course similar to the disappearance in the ovule.
Our data suggest that RGA degradation in fertilized ovules
during fruit-set is most probably due to elevated GA levels,
achieved by auxin-induced upregulation of GA biosynthesis
genes after fertilization. Interestingly, RGA degradation is
ovule/seed autonomous (Figure 9), suggesting that no GA
diffusion occurs from fertilized to unfertilized ovules. In
addition, degradation of RGA in the valves indicates the
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
326 Eavan Dorcey et al.
(a)
(b)
30
AtGA20ox1
AtGA20ox1
6
20
4
10
2
0
4
Relative gene expression level
Relative gene expression level
1
AtGA20ox2
3
2
1
0
8
0
16
AtGA20ox2
12
8
4
0
AtGA3ox1
AtGA3ox1
12
6
8
4
4
2
1
0
0
2
6 24 48
GA3
2
6 24 48
NPA
2
6 24 48 h
2,4-D
P V O
Mock
P V O
P V O
P V O
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
(Figure 6) does not support upregulation of GA synthesis in
the valves, a likely possibility is that they are translocated
from the fertilized ovules, where they are synthesized
according to our gene expression data.
olism specifically in fertilized ovules; (iii) GA signaling is
detected in both seeds and valves; and (iv) constitutive GA
signaling is sufficient to promote parthenocarpic fruit
development.
Discussion
Auxin response in the fertilized ovule is an early
event during fruit-set
Our analysis of the molecular events associated with the
action of auxin and GA during fruit-set indicates that:
(i) auxin response in young seeds is an early event during
fruit-set; (ii) auxin promotes the activation of GA metab-
Through the analysis of ProDR5rev:GFP transgenic plants,
we have shown that an auxin response is activated in ovules
soon after fertilization, probably as a consequence of
elevated auxin levels. Several explanations could account
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 327
Figure 9. Gibberellin (GA) signaling occurs in
both ovules and valves upon fruit-set.
Pistils of cer6-2 plants harboring the fusion
protein GFP-RGA under the control of the endogenous promoter RGA (Silverstone et al., 2001)
were either treated with mock solution, GA3, N-1naphthylphthalamic acid (NPA), 2,4-dichlorophenoxyacetic acid (2,4-D), or hand-pollinated
with Landsberg erecta (Ler) pollen. Images were
taken 24 and 48 h later, and are the composite of
fluorescent GFP and bright field images. Chlorophyll fluorescence is observed as a red signal,
while GFP appears as green signal. u, unfertilized
ovule; f, fertilized ovule.
for this observation: a local increase in auxin synthesis,
a blockage of auxin transport, interference with auxin
response upon fertilization, or even the downloading of
auxins from the pollen tube into the ovule (Sweet and Lewis,
1969). However, the fact that NPA treatment induces parthenocarpic fruit development by mimicking the same auxin
response in the ovule seen upon fertilization rules out the
latter two possibilities. Parthenocarpic development
induced by a block in polar auxin transport was previously
reported in cucumber (Robinson et al., 1971; Beyer and
Quebedeaux, 1974), and it was proposed that the effect
might be due to a blockade in the outward flow of auxin from
the ovary, resulting in auxin accumulation sufficient to
trigger fruit-set in absence of fertilization.
Whether the increase in auxin in the fertilized ovule is
due to enhanced biosynthesis or interference in transport
cannot be determined based on our results. Nevertheless,
we have not observed any modification in the abundance
and localization of several PIN auxin efflux carriers (Blakeslee et al., 2005) during pistil development and fertilization
(data not shown). Thus, it seems likely that auxin synthesis
is activated in the ovule early after fertilization and that this
may initiate fruit development. Several pieces of evidence
support this view. First, auxins are naturally present in
developing ovules. We have detected a very low GFP signal
driven by the ProDR5 in mock-treated ovules, which indicates that a basal level of auxin is present in unfertilized
ovules. In addition, conjugated auxins have been detected
in the ovules of unfertilized pistils (Aloni et al., 2006) and in
developing ovules and embryos (Benkova et al., 2003; Friml
et al., 2003). Second, exogenous auxin can induce parthenocarpy in a variety of species, including Arabidopsis.
Finally, overexpressing IAA-biosynthesis genes in ovules or
placenta of transgenic plants result in parthenocarpy in
several species (for example, Rotino et al., 1997; Yin et al.,
2006). In summary, either application of auxin or overexpression of auxin biosynthetic genes in ovules would
promote the auxin response observed upon fertilization. It
is then plausible to hypothesize that elevated levels of auxin
in the ovule would initiate a cascade of events that would
finally promote fruit growth. These events would include
auxin signaling, through the function of Aux/IAA and ARF
proteins (Swain and Koltunow, 2006; Pandolfini et al.,
2007), and crosstalk with other hormones (Weiss and Ori,
2007).
Auxin action is mediated by gibberellins in the ovary
Although the efficiency of the different hormones in the
induction of fruit development varies between species, the
activation of GA metabolism after fertilization is a common
theme. For instance, early fruit development in tomato
plants is characterized by elevated mRNA levels of copalyl
diphosphate synthase, a gene involved in the early steps of
GA biosynthesis, LeGA20ox1 and LeGA20ox3, accompanied
by decreased level of LeGA3ox2 (Rebers et al., 1999). Indeed,
the expression of GA 20-oxidase genes seems to be a limiting step for the increase in GA concentration during fruitset (Serrani et al., 2007). A similar trend has been observed
in pea, where a GA 20-oxidase gene is strongly upregulated
during early fruit development, both in the developing seeds
and in the pod (Garcia-Martinez et al., 1997).
How does fertilization activate GA biosynthesis? Our
results suggest that the initial event that causes this
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
328 Eavan Dorcey et al.
increase in GA concentration may be the activation of auxin
signaling in the ovules. Again, this connection is not
species-specific, and the particular steps in GA metabolism
affected by auxin are equivalent between species. For
instance, in pea the auxin 4-Cl-IAA induces expression of
GA biosynthesis genes (PsGA20ox1 and PsGA3ox1) and
accumulation of GAs (Van Huizen et al., 1995, 1997; Ozga
and Reinecke, 1999; Ozga et al., 2003), which is in agreement with our observations that auxin upregulates AtGA20ox1, AtGA20ox2, AtGA20ox3, and AtGA3ox1 in early
fruit development in Arabidopsis. Furthermore, auxin
induces fruit-set and growth in tomato, at least partially,
by enhancing GA biosynthesis and decreasing GA inactivation activity, leading to a higher bioactive GA content
(Serrani et al., 2008). However, we cannot rule out the
existence of a parallel pathway where GA biosynthesis
would be activated directly after fertilization, in an auxinindependent manner.
Interestingly, GA application triggers fruit development
without any alteration in auxin response, based on the
analysis of ProDR5rev:GFP expression. These results point
to a hierarchy in the action of these hormones, according to
which fruit development would be initiated after fertilization
by the sequential action of auxins and GAs. If this is the case,
the co-occurrence of GA biosynthesis and signaling would
be an absolute requirement for fruit development, induced
either by fertilization or by auxin treatments. Several pieces
of evidence support this hypothesis. First, double loss-offunction of AtGA20ox1 and AtGA20ox2, or ectopic expression of a GA 2-oxidase gene, causes seed abortion and
shorter siliques in Arabidopsis (Singh et al., 2002; Rieu et al.,
2008b). Second, the use of inhibitors of GA biosynthesis
hinders auxin-induced fruit-set in tomato, an effect that is
reversed by GAs, suggesting that the effect of auxin is fully
mediated by GA (Serrani et al., 2008). Third, expression in
tomato of a gain-of-function allele of an Arabidopsis DELLA
protein, gai-1D, reduces fruit development (Marti et al.,
2007). Fourth, silencing of the single tomato DELLA gene
(SlDELLA) results in the formation of parthenocarpic fruits in
the absence of pollination (Marti et al., 2007), indicating that,
at least in tomato, fruit initiation is mediated by DELLA
proteins. The only observation that seems to contradict this
hypothesis is the ability of auxin to induce fruit-set in the
Arabidopsis gai-1D mutant (Vivian-Smith and Koltunow,
1999), although redundancy of DELLA proteins in Arabidopsis might account for this effect, i.e. auxin-induced fruit-set
would be mediated not by GAI but by RGA or RGL proteins.
However, it is also possible that auxins and GAs have
specific independent roles in the promotion of fruit development, beyond the early and sequential action described
above. Support for this scenario comes from the observation
that only GAs promote certain differentiation processes
early during fruit development, such as the collapse of the
ena layer.
Coordinated hormone signaling in ovules and valves
Upon ovule fertilization, the initiation of embryo development triggers elongation of the pistil to host the developing
seeds. Thus, a growth-induction signal has to be generated
to promote growth of the valves. Most probably, this
signaling molecule would be hormonal in nature, but where
this molecule is generated and how it is transported is
unknown. Our data indicate that: (i) fertilization-induced
changes in auxin response are detected in young seeds, but
not in valves; (ii) GA metabolism is activated upon fertilization or during auxin-induced fruit development, specifically
in seeds, at least within the first 72 h upon pollination; and
(iii) changes in GA signaling are detected in both seeds and
valves. In this scenario, it is likely that auxin present in
fertilized ovules promotes GA synthesis specifically in the
young seeds, which suggests that there might be a
GA-specific regulatory function in seed development.
Furthermore, we have shown that the different genes
involved in GA biosynthesis act in a sequential manner:
some genes are upregulated early upon fruit-set while
others are activated later on, the latter probably being
associated with seed development (Figure 10). The GAs
synthesized in the seed would then be transported to valves
to promote GA signaling and finally to promote coordinated growth. In other words, GAs would be necessary for
both seed and valve development. In this regard, a close
correlation between seed development and pod elongation
has been reported in Arabidopsis, which implies that a
seed-derived growth-promoting signal would be transferred through the funiculus to the adjacent valve structures
and allow growth (Cox and Swain, 2006). It has been shown
that AtGA20ox2 makes the greater contribution to fruit
elongation, as its loss-of-function provokes shorter siliques,
but with similar seed numbers, than in wild-type plants
(Rieu et al., 2008b). A similar effect was observed for the
loss-of-function of AtGA3ox1 (Hu et al., 2008). Since this
phenotype could not be fully rescued by pollination with
wild-type pollen, it was postulated that the GAs regulating
silique elongation would be of maternal origin, i.e. that GAs
would probably be synthesized (via AtGA20ox2 and
AtGA3ox1 activity) in the silique tissue itself or in the seed
endosperm (Hu et al., 2008; Rieu et al., 2008b). Based on
our observation that the expression of these genes is
localized in seeds but not in silique walls, it is possible that
the maternal effect described above could be attributed to
GAs being synthesized in the endosperm, but not in the
valve tissue. The loss-of-function of AtGA3ox4, in combination with ga3ox1, results in shorter fruits that can be
rescued by wild-type pollen, suggesting an embryo- or
seed-effect (Kim et al., 2005; Hu et al., 2008). Nevertheless,
the observation that a quintuple GA 2-oxidase lossof-function mutant, in which biosynthesis of bioactive GA
is unaltered but inactivation is blocked, develops
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 329
Temporal pattern
Spatial pattern
Time (h) 0
24
48
72
GA20ox1
GA20ox2
GA20ox3
GA20ox4
GA20ox5
GA3ox1
GA3ox2
GA3ox3
GA3ox4
GA2ox1
GA2ox2
GA2ox3
GA2ox4
GA2ox6
GA2ox7
GA2ox8
Experimental procedures
Ovule
GA20ox1
GA20ox2
GA20ox3
GA20ox4
GA20ox5
be to promote fruit growth and cellular differentiation,
including cell expansion and degradation of the inner
endocarp cell layer. The collapse of ena occurs naturally
towards the end of stage 17 of pollinated fruit development
(Ferrandiz et al., 1999; Roeder and Yanofsky, 2006), as well
as in auxin-induced fruits (our data), but seems to be
accelerated in GA-induced fruits. Similar data were reported
in emasculated spy-4 pistils induced to grow with GA3, but
not in wild-type pistils (Vivian-Smith and Koltunow, 1999).
These data suggest that degradation of ena prior to pod
shattering is controlled by GAs, as is the case for
other developmental processes that take place during fruit
development, such as cell expansion in mesocarp cells
(Vivian-Smith and Koltunow, 1999).
Valve
GA3ox1
GA3ox2
GA3ox3
GA3ox4
GA2ox1
GA2ox2
GA2ox3
GA2ox4
GA2ox6
GA2ox7
GA2ox8
Figure 10. Schematic representation of the temporal and spatial expression
of gibberellin (GA) metabolism genes upon fruit-set.
Arrows indicate the temporal expression frame of the corresponding gene,
between anthesis and 72 h after fertilization, in the ovule (upper panel) and
valve (lower panel).
parthenocarpic siliques (Rieu et al., 2008a) implies that
even in the absence of fertilization the pistil must have
access to GAs that would then accumulate and drive
parthenocarpic fruit development. Our results suggest that
these active GAs would have to be transported to the
silique from other tissues, but specific experiments would
be needed to address this question.
Finally, our data show that while GA synthesis occurs only
in the young seed (Figure 10), GA signaling is detected both
in developing seeds and in valves, indicating that GAs could
be the hypothesized growth-regulator that coordinates seed
and fruit development. The role of GAs in the valves would
Plant material and hormone treatments
Arabidopsis thaliana cer6-2 seeds, in the Landsberg erecta (Ler)
accession (Preuss et al., 1993; Fiebig et al., 2000), were obtained
from the Arabidopsis Biological Resource Center (ABRC). Seeds
from the lines ProDR5rev:GFP in the Columbia-0 accession (Col-0)
(Benkova et al., 2003), the ProRGA:GFP-RGA in Ler (Silverstone
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
(Tübingen University, Germany), Tai-ping Sun (Duke University,
NC, USA), and Nicholas P. Harberd (John Innes Centre, Norwich,
UK), respectively.
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
cer6-2. For each plant, only flowers from the primary bolt, between 0
and 2 days post-anthesis (dpa) were used. For plants not harboring
the mutant cer6-2 allele (i.e. the parental Ler, the transgenic line
harboring the ProDR5rev:GFP construct, and the quadruple-DELLA
mutant), self-pollination was avoided by emasculation 1 day before
anthesis. Fruit-set was induced by hand pollination with Ler pollen
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,
http://www.duchefa.com/). These concentrations were tested to be
optimal to induce fruit-set. Tween-80 at 0.05% was used as the
wetting agent, and the pH was adjusted between 6.5 and 7.0.
Samples were harvested and processed at the indicated timepoints. For dissection experiments, valves and ovules were collected by hand dissection under a stereoscope microscope with the
help of a razorblade and acupuncture needles, placed in a vial with
RNA extraction buffer, and RNA was immediately extracted (see
below).
To test induction of fruit-set, pistil or fruit length was measured at
7 dpa or at full maturity. Pistils and fruits were harvested and
scanned, and images were analyzed using Image J software
(Abramoff et al., 2004).
Scanning electron microscopy (SEM)
Samples were harvested, mounted on the specimen holder of a CT1000C cryo-transfer system (Oxford Instruments, http://www.
oxford-instruments.com/), interfaced with a JEOL JSM-5410
scanning electron microscope, and frozen in liquid N2. Samples
ª 2009 The Authors
Journal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 58, 318–332
330 Eavan Dorcey et al.
were fractured, and sublimated by controlled heating at )85C.
Finally, samples were observed at incident electron energy of
10 keV with 10 · to 100 · magnification.
Confocal microscopy
A Leica TCS SL confocal microscope (Leica Microsystems, http://
www.leica-microsystems.com/) was used. Green fluorescent protein was excited at 488 nm and emission was detected between 500
and 520 nm. Endogenous chlorophyll was excited with the same
wavelength but detected between 660 and 690 nm. The identity or
specificity of each signal was confirmed with a k-scan.
Quantitative RT-PCR analysis of gene expression
Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen,
http://www.qiagen.com/). Genomic DNA was eliminated by treatment with 50 units of DNaseI (RNase-free DNase set; Qiagen) for
15 min at room temperature. Two micrograms of total RNA were
used to synthesize first-strand cDNA, using the SuperScript FirstStrand Synthesis System for RT-PCR (Invitrogen Life Technologies,
http://www.invitrogen.com/). The cDNA synthesis reactions were
finally diluted in a volume of 80 ll.
Quantitative RT-PCR was carried out using the SYBR GREEN
PCR Master Mix (Applied Biosystems, http://www3.appliedbiosystems.com/) in an ABI PRISM 7000 Sequence Detection System
(Applied Biosystems). The final reaction volume was 20 ll, with 1 ll
of cDNA, 10 ll of SYBR GREEN PCR Master Mix, and 9 ll of primer
mixture, containing 0.66 lM of each primer. The PCR program
consisted of an initial incubation of 2 min at 50C followed by a
denaturation at 95C for 10 min, and 40 cycles of amplification of 15
sec at 95C and 1 min at 60C. In a single experiment, each sample
was assayed in triplicate. Expression levels were calculated relative
to the constitutively expressed genes ACT8 or phosphatase 2A
(PP2A) subunit PDF2 (At1g13320) (Czechowski et al., 2005), which
were tested to be constitutive in the different tissues used in each
experiment (PP2A for whole pistils and fruits, and ACT8 for
dissected ovules and valves). Normalization was carried out using
the DDCt method (Applied Biosystems), where DCt was calculated
for each sample as the difference between Ct (gene of interest) and
Ct (constitutive gene), and the final relative expression level was
determined as inverse of log2 of Ct (sample) – Ct (reference sample).
Normalization was as indicated in the figure legends and in the text.
Primer sequences for amplification have been previously described
(Curaba et al., 2004; Czechowski et al., 2005; Frigerio et al., 2006)
and are shown in Table S1. All experiments were repeated twice,
with similar results.
Acknowledgements
We thank Jiri Friml (Tübingen University, Germany), Tai-ping Sun
(Duke University, NC, USA), and Nicholas P. Harberd for the gift of
ProDR5rev:GFP, ProRGA:GFP-RGA, and quadruple–DELLA mutant
seeds, respectively; J. L. Garcia-Martinez and D. Alabadi for critical
reading of the manuscript; and Ms M. A. Argomániz for excellent
technical assistance. ED was supported by an FPI Fellowship from
the Spanish Ministry of Education and Science (MEC). MAPA
received a post-doctoral contract from the ‘Ramón y Cajal’ program
from MEC. MAB was supported by the EMBO Young Investigator
Program. This work was funded by grants from the Spanish MEC
BIO2002-04083-C03-02 and BIO2005-07156-C02-01.
Supporting Information
Additional Supporting Information may be found in the online
version of this article:
Table S1. List of the genes of gibberellin (GA) metabolism and
oligos used for quantitative RT-PCR.
Figure S1. Expression of genes involved in gibberellin (GA) metabolism in pistils of cer6-2 plants at anthesis.
Figure S2. Expression of gibberellin (GA) 20-oxidase genes in fruits
of cer6-2 after treatment with GA3, N-1-naphthylphthalamic acid
(NPA) or 2,4-dichlorophenoxyacetic acid (2,4-D).
Figure S3. Expression of gibberellin (GA) 3-oxidase genes in fruits of
cer6-2 after treatment with GA3, N-1-naphthylphthalamic acid (NPA)
or 2,4-dichlorophenoxyacetic acid (2,4-D).
Figure S4. Expression of gibberellin (GA) 2-oxidase genes in fruits of
cer6-2 after treatment with GA3, N-1-naphthylphthalamic acid (NPA)
or 2,4-dichlorophenoxyacetic acid (2,4-D).
Figure S5. Expression of gibberellin (GA) 20-oxidase genes in
dissected fruits of cer6-2 after 6 h of treatment with GA3, N-1naphthylphthalamic acid (NPA) or 2,4-dichlorophenoxyacetic acid
(2,4-D).
Figure S6. Expression of gibberellin (GA) 3-oxidase genes in
dissected fruits of cer6-2 after 6 h of a treatment with GA3, N-1naphthylphthalamic acid (NPA) or 2,4-dichlorophenoxyacetic acid
(2,4-D).
Figure S7. Expression of gibberellin (GA) 2-oxidase genes in
dissected fruits of cer6-2 after 6 h of a treatment with GA3, N-1naphthylphthalamic acid (NPA) or 2,4-dichlorophenoxyacetic acid
(2,4-D).
Please note: Wiley-Blackwell are not responsible for the content or
functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
corresponding author for the article.
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