Institut des Sciences du Végétal, UPR2355 Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91190 Gif-sur-Yvette, France
Author for correspondence (e-mail:
francois.parcy{at}isv.cnrs-gif.fr)
Accepted 4 August 2003
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SUMMARY |
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Key words: Seed maturation, Transcriptional regulation, Storage protein, ABI3, FUS3, LEC2
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Introduction |
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We combined the information obtained from the exhaustive characterisation of the napA promoter and the power of Arabidopsis genetics to study the regulation of the At2S3 promoter in Arabidopsis, which is very similar to napA promoter. We initiated a one-hybrid screen in yeast to identify transcription factors able to interact with the different cis-regulatory elements. This screen yielded the two B3 factors FUS3 and LEC2 but not ABI3. This result led us to investigate the respective roles of all 3 homologous transcription factors in planta. We propose that ABI3 regulates At2S3 expression indirectly and that FUS3 and LEC2 regulate At2S3 directly in a partially redundant manner. The extent of this redundancy is determined by the overlapping but not identical expression patterns of FUS3 and LEC2 and by a so far unsuspected regulation of FUS3 by LEC2.
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Materials and methods |
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Yeast one-hybrid experiments
cDNA library
RNA was extracted from 8 g of 5-12 DAP (days after pollination) Col-0
siliques using a phenol-chloroform extraction
(Parcy et al., 1994) followed
by a clean-up with RNeasy midi kit (Qiagen, Hilden, Germany). After mRNA
purification using an mRNA purification kit (Amersham-Pharmacia, Little
Chalfont, UK), the cDNA library was built in the pAD-GAL4-2.1 vector with
HybriZAP-2.1 XR cDNA synthesis kit and HybriZAP-2.1 XR library construction
kit (Stratagene, La Jolla, USA). The cDNA library represented
7x106 independent cDNA clones.
Yeast reporter strains
The reporter constructs were made in the plasmid pYi2267OHIS
(Blaiseau et al., 1997), which
carries the URA3 selection marker and the minimal CYC1
promoter upstream of the HIS3 reporter gene. The region 170 to
45 of the Col-0 At2S3 promoter (relative to the transcription
start), the RY-G-box complex (95 to 48)
(5'-ATCACTCATGCATGCATGCATTCTTACACGTGATTGCCATGCAAATCTCCC-3') and
the B-box (169 to 132)
(5'-ATCTGTTCGTCACTTGTCACTCTTTTCCAACACATAATCCC-3') were cloned into
the XhoI site of pYi2267OHIS, giving rise to the plasmids pYi22-1,
pYi22-G and pYi22-B, respectively. These plasmids and the empty plasmid
pYi2267OHIS were linearised and transformed into the yeast strain YM954
(Blaiseau et al., 1997
)
generating the strains At2S3::HIS3, RY-G::HIS3, B::HIS3 and Yi22-C,
respectively. Stable prototroph transformants were selected on SD medium
(Ausubel et al., 1989
) lacking
uracil, and analysed for correct integration by PCR analysis.
One-hybrid screen
The At2S3::HIS strain was transformed with the hybrid expression library,
and colonies growing on SD medium Leu, His, +10 mM
3-aminotriazol (3-AT) were isolated. Plasmid DNA was recovered from these
colonies and retransformed into At2S3::HIS3 and Yi22-C. Transformants were
selected on SD medium Leu and then tested on SD medium Leu,
His, +10 mM 3-AT.
One-hybrid analysis
LEC2 and FUS3 coding sequences were cloned into pCV70, a
derivative of pRS315 (Sikorski and Hieter,
1989), which allows expression of HA-tagged proteins under the
control of the ADH promoter. For GAL4-AD fusions, LEC2, FUS3
and ABI3 coding sequences were cloned in pDON201 and recombined in
pDEST22-PC86 using the Gateway technology (Invitrogen, La Jolla, USA). The
fusion between the VP16 activation domain
(Parcy et al., 1998
), a short
ABI3 N-terminal piece (amino acids 3-13) and ABI3 B3 domain (aa 556-720) did
not lead to a detectable activation of an At2S3::LACZ reporter
construct built in the pKF1 vector (Parcy
et al., 1998
).
Electrophoretic mobility shift assays (EMSA)
EMSA were performed essentially as previously described
(Bensmihen et al., 2002).
FUS3 and LEC2 coding sequences were PCR amplified using
oligonucleotides 5'-ATGATATCCATGGTTGATGAAAATGTGGAAACC-3' and
5'-ATGATATCTAGTAGAAGTCATCGAGAG-3' for FUS3 and
5'-TCTAGAAAAATGGATAACTTCTTACCCT-3' and
5'-GTCGACCCATATCACCACCACTCAAAGT-3' for LEC2, cloned into
PCRT7/CT-TOPO (Invitrogen) and sequenced. The resulting plasmids were used for
in vitro transcription and translation in TNT®-rabbit reticulocyte system
(Promega, Madison, USA). Probes and competitor DNA was obtained by annealing
the following oligonucleotides (mutant bases are underlined):
Generation of transgenic plants
All transgenic plants were obtained by floral dip of Arabidopsis
Col-0 (Clough and Bent, 1998).
Plasmids were built according to standard molecular biology procedures
(Ausubel et al., 1989
).
At2S3::GFP plasmid (pFP91) was built by assembling At2S3
promoter fragment (310 to +35 relative to the transcription start) to
an optimised green fluorescent protein (GFP) coding sequence with a
translational enhancer and an endoplasmic reticulum targeting signal (obtained
from R. Blanvillain and P. Gallois) and the 35S terminator. Over 60
independent lines were generated and one representative line (FP91.54.3) was
chosen for subsequent analyses. LEC2::GUS plasmid (pTK-DE111) was built by
inserting the LEC2 promoter (2020 to +5 relative to ATG of
LEC2) into pDE-GUS vector (Parcy
et al., 1994
). Three independent lines were generated that showed
the same expression profile. The FUS3::GUS reporter plasmid was generated by
inserting the FUS3 promoter (2100 to +44 relative to ATG of
FUS3) into pDE-GUS. 12 plants were generated of which 11 showed a
similar expression profile.
Expression analyses
Northern blot analysis was performed as described previously
(Parcy et al., 1997).
Gene-specific probes were PCR amplified from genomic DNA with the following
oligonucleotides: 5'-CTTCAACATCCCTTCATTCCCTT-3' and
5'-TCTTATTTATTAAGTAGTGCTT-3' (At2S1),
5'-TCCAGACCACCATCCCTTTCTT-3' and
5'-GACAACCTAGAGAGAGCATA-3' (At2S2),
5'-TTCCAGATCCCTTCAATCCCTT-3' and
5'-AACATAAACAAACCTCTCTTA-3' (At2S4),
5'GCCGCCTTTGAGGGGCCAGA-3' and
5'-CCTTGTGGTACGGCTATGAG-3' (CRA),
5'-CACCCTTGAGACGCGGCGAA-3' and
5'CCTTGTGGCACGACTAGTAA-3' (CRB) and
5'-AGACCTTCATGGACTCGCAG-3' and
5'-GCATGTCACGGAACCCTTGTTG-3' (CRC).
At2S3-specific probe was obtained by subcloning a fragment
corresponding to nucleotides 536 to 690 from gene At4G27160.1 into PCRII-TOPO
(Invitrogen, La Jolla) generating pTOPAT2S3. RT-PCR analysis was performed on
silique total RNAs extracted using the RNeasy mini kit (Qiagen). The following
oligonucleotides were used for PCR amplification:
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Results |
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ABI3, FUS3 and LEC2 regulate At2S3
expression differently in planta
The results of our yeast experiments, which suggested a direct and similar
role for FUS3 and LEC2 and an indirect role for
ABI3 in regulating At2S3, prompted us to analyse their
respective roles in planta. We analysed At2S3 regulation in severe
abi3, fus3 and lec2 mutant alleles. We used the
abi3-6 mutant allele that was previously shown to contain a large
deletion at the ABI3 locus
(Nambara et al., 1994). We
sequenced this locus and found that the C terminus of the ABI3-6
coding sequence is not in frame with the short N-terminus. Abi3-6 is
thus the only abi3 allele that lacks the three B1, B2 and B3 domains.
We used the null lec2-1 allele in the Ws background
(Stone et al., 2001
). We
observed, based on the anthocyanin accumulation patterns in mutant seeds, that
the phenotype of lec2-1 seeds is extremely variable from seed to
seed. The same was true for lec2-2, -3 and -4 (data
not shown). We used fus3-3 in the Col-0 background
(Luerssen et al., 1998
) which
also shows a phenotype of variable intensity. Unless otherwise indicated,
abi3, lec2 and fus3 will, from now on, refer to abi3-6,
lec2-1 and fus3-3, respectively.
In order to easily follow the temporal and spatial patterns of
At2S3 activation, we built a transgenic Arabidopsis line
carrying a fusion between the At2S3 promoter and the coding sequence
of the GFP, and crossed this At2S3::GFP line to the abi3,
lec2 and fus3 mutants. We first used northern blot analysis to
measure the quantitative effects of all 3 mutations on At2S3 and
At2S3::GFP mRNA levels (Fig.
2A). We found that At2S3 and GFP expressions
were most reduced in abi3 (over 60 fold) and to a lesser extent in
fus3 (8-9 fold) and lec2 (1.5-2.5 fold)
(Fig. 2 and data not shown).
Because each of the mutations reduced the At2S3 and GFP mRNA
levels by a similar factor, we concluded that all three B3 factors reduced
At2S3 mRNA steady-state level by affecting the At2S3
promoter activity. We used the At2S3::GFP line to determine how the
three different mutations differentially affect the spatial expression of
At2S3. In the wild-type background, At2S3::GFP fluorescence
started at torpedo stage in the embryo axis, spread in the whole embryo at the
end cotyledon stage and ended up stronger in cotyledons than in the axis of
the dry seed (16-18 DAP) (data not shown and
Fig. 3I). In addition,
fluorescence was absent from the root meristem and detectable in the endosperm
layer of the dry seed. This dynamic expression pattern is similar to what has
been described for Brassica napus SSP genes
(Fernandez et al., 1991). In
abi3 mutant embryos, At2S3::GFP fluorescence was
consistently and strongly reduced as compared to wild-type embryos
(Fig. 3B,F). It was detected at
low levels in the embryo axis and in the centre of cotyledons and undetectable
in the endosperm (Fig. 3J,K,T).
Fus3 and lec2 mutations lead to very variable phenotypes
(Fig. 3C,D,G,H). In the
lec2 mutant, the fluorescence was often slightly reduced throughout
the embryo but the most striking phenotype was the presence of sectors
accumulating anthocyanins where the fluorescence was totally absent
(Fig. 3N-R). These sectors
varied in shape and size between embryos from the same silique and between
cotyledons of the same embryo (Fig.
3O,Q,R). In mature lec2 seeds, endosperm fluorescence was
not reduced (Fig. 3U).
At2S3::GFP fluorescence was also reduced in fus3 mutant
embryos. This reduction was sometimes very mild, making fus3 seeds
hardly distinguishable from wild types (data not shown) while in the most
severe fus3 embryos, the fluorescence was completely absent at the
cotyledon periphery, very weak in the cotyledon centre and reduced in the
embryo axis (Fig. 3L,M).
Endosperm fluorescence was almost abolished in fus3 mutants
(Fig. 3V). Our analyses of the
At2S3::GFP transgenic line were essentially confirmed by in situ
hybridisation showing that the At2S3::GFP reporter faithfully
reproduced At2S3 expression. At2S3 mRNA was undetectable in
abi3 embryos (Fig.
2B-4) and fus3 cotyledons
(Fig. 2B-2). It was either
undetectable or reduced in fus3 axis and in discrete regions of
lec2 cotyledons (Fig. 2B-3, -5,
-6). Expression in the endosperm layer, which was more difficult
to detect with confidence, was often observed in Col-0 and lec2 but
always absent from abi3 and fus3 endosperm
(Fig. 2B-7 to 2B-10). In
summary, our analysis of At2S3 expression in seeds showed that all
three B3 regulators are important but to different extents: ABI3 has
a major role in regulating At2S3, whereas, FUS3 and
LEC2 appear to be dispensable in some parts of the embryo.
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Discussion |
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Several lines of evidence indicate that ABI3 regulates
At2S3 expression by a different mechanism than LEC2 and
FUS3. First, the ABI3 protein present in the lec2 fus3
double mutant is unable to compensate for the loss of FUS3 and
LEC2 functions, indicating that, despite its B3 and activation
domains, ABI3 is not sufficient to activate At2S3. In agreement with
this conclusion, we observed that transformation of lec2 fus3
At2S3::GFP plants with a 35S::ABI3 construct did not yield any
fluorescent seed, whereas transformations with 35S::LEC2 or
35S::FUS3 did (G.S. and F.P., unpublished). Similarly, we observed
using RT-PCR analysis that FUS3 and LEC2 are normally
expressed in abi3 mutant seeds (data not shown) and yet unable to
compensate for the lack of ABI3 and fully induce At2S3. However, FUS3
and LEC2 are not completely inactive in the absence of ABI3 since they are
responsible for the low At2S3 expression level present in
abi3 mutant (F.P., unpublished). Based on these results and on
previous studies on the regulation of the At2S and napA
promoters (Ezcurra et al.,
2000; Lara et al.,
2003
) we propose a speculative model
(Fig. 6B), which predicts that
the activation of At2S3 would require three types of proteins: ABI3,
FUS3/LEC2 and bZIP10/bZIP25. While, according to the model, only FUS3/LEC2 and
the bZIPs interact directly with the RY-G-box complex, ABI3 is tethered to the
promoter through interactions with the bZIPs. FUS3/LEC2 are necessary for
At2S3 activation. When the FUS3/LEC2 binding sites are mutated
(Ezcurra et al., 2000
), or in
the lec2 fus3 double mutant, At2S3 expression is abolished.
When the G-box motif is mutated, so that the bZIP proteins cannot recruit ABI3
to the RY-G-box complex or when ABI3 itself is inactivated, transactivation of
the promoter is drastically reduced. Nevertheless, FUS3 and LEC2 alone are
sufficient to slightly activate At2S3 even in the absence of ABI3 as
observed in yeast or in the abi3 mutant. Other SSP genes seem to be
regulated via the same mechanisms as At2S3 since their expressions
levels in the lec2 fus3 double mutant are also drastically reduced
when compared to wild type. Their regulation might, however, differ slightly
from one SSP gene to another because the effects of single mutations on
individual genes are not identical. At2S1, for example, is less
affected by fus3 or lec2 single mutations than other
At2S genes, probably because At2S1 is specifically expressed
in the embryonic axis (Guerche et al.,
1990
) where the effects of the fus3 and lec2
single mutations are the weakest (as judged by At2S3 expression).
FUS3 and LEC2 expression patterns explain
lec2 and fus3 mutant phenotypes
If the FUS3 and LEC2 proteins are indeed functionally equivalent and if
they are both expressed throughout the embryo, we would predict that they
regulate At2S3 in a completely redundant manner. The local lack of
At2S3 expression in lec2 or in fus3 single mutants,
however, indicated that this is not the case. Indeed, we have shown that
phenotypes of fus3 and lec2 single mutants can be explained
by non-uniform FUS3 and LEC2 expression patterns. From 12
DAP on, LEC2::GUS expression was absent from the periphery of the
cotyledons. This pattern was strikingly similar to the At2S3::GFP
expression pattern in fus3 embryos of the same age, as one would
expect if At2S3 expression totally depends on LEC2. We thus propose
that LEC2 transcription is a limiting factor for At2S3
expression in the fus3 background. However, we do not yet understand
why the fus3 phenotype is variable and why some dry fus3
mutant seeds still bear a high level of fluorescence. As opposed to LEC2,
FUS3 is uniformly expressed throughout the wild-type embryo. This
expression pattern was difficult to reconcile with the lack of At2S3
expression in sectors of lec2 mutant embryos. We have solved this
apparent paradox by showing that FUS3::GUS activity becomes very heterogeneous
in a lec2 mutant and is absent wherever At2S3 expression is
missing. The absence of both essential factors (LEC2 is inactive and
FUS3 not expressed) explains why At2S3 expression is
abolished in these sectors. The precise coincidence between FUS3::GUS activity
and At2S3 expression strongly suggests that FUS3 expression
is the limiting factor for At2S3 expression in lec2. In
support of this conclusion, transformation of lec2 mutants with a
35S::FUS3 construct almost completely suppresses the presence of
sectors devoid of At2S3::GFP fluorescence (G.S. and F.P.,
unpublished). Our analysis of FUS3 and LEC2 promoter
activities are consistent with RT-PCR experiments
(Fig. 5R) and the expression of
the At2S3 target gene in fus3 or lec2 mutants. For
these reasons, we think FUS3::GUS and LEC2::GUS activities faithfully reflect
the expression of FUS3 and LEC2 genes. However, the
confirmation of the precise expression patterns of FUS3 and
LEC2 will require immunolocalization or in situ hybridisation.
In the endosperm, FUS3 and LEC2 expression patterns also
provide an explanation for the fus3 and lec2 phenotypes. In
the mature endosperm, we have detected FUS3::GUS and ABI3::GUS activities
(Parcy et al., 1994) but no
LEC2::GUS activity. In agreement with these expression profiles,
At2S3 expression is almost abolished in fus3 or
abi3 but unaffected in lec2. Our model
(Fig. 6) therefore also applies
for At2S3 expression in the endosperm with FUS3 and ABI3 as major
actors. In conclusion, we think that FUS3 and LEC2
expression patterns and their functional similarity explain At2S3
expression in lec2 and fus3 mutants. However, it is likely
that FUS3 and LEC2 are not completely interchangeable. We have, for example,
observed that a 35S::LEC2 construct induces ectopic At2S3
expression in leaf or floral tissue while a 35S::FUS3 construct does
not (T.K., G.S. and F.P., unpublished).
Implications of FUS3 regulation by LEC2 for seed
maturation
Our analysis of FUS3::GUS activity showed that LEC2 controls
FUS3 expression. However, since FUS3 expression is not
abolished in lec2 mutant, other factors must be involved in
FUS3 activation. The variability of the lec2 phenotype is
probably due to variable activation of FUS3 by this unknown factor.
The observation that LEC2 regulates FUS3 has implications
for seed maturation in general: the existence and the nature of interactions
between the four major regulators of maturation (ABI3, FUS3, LEAFY COTYLEDON1
and LEC2) has often been raised but never explained
(Bäumlein et al., 1994;
Keith et al., 1994
;
Meinke, 1992
;
Meinke et al., 1994
;
Parcy et al., 1997
;
Raz et al., 2001
;
Vicient et al., 2000
).
FUS3 regulation by LEC2 is the first demonstrated
interaction between two of these regulators. It is likely that the lack of
FUS3 expression in sectors of lec2 cotyledons is responsible
for other lec2 phenotypes such as anthocyanin accumulation. In
agreement with this assumption, constitutive expression of FUS3 in
lec2 almost abolishes anthocyanin accumulation (G.S. and F.P.,
unpublished). The lec1 mutation also results in a reduced expression
of the SSP gene (Parcy et al.,
1997
; Vicient et al.,
2000
). The nature of the interactions between LEC1 and
ABI3, LEC2 and FUS3 has never been clearly elucidated.
According to available data, lec1 is unlikely to completely abolish
the expression of one of the three B3 genes
(Meinke et al., 1994
;
Parcy et al., 1997
;
Raz et al., 2001
;
Stone et al., 2001
;
Vicient et al., 2000
).
However, as suggested by the effect of lec2 on FUS3
expression, it is possible that LEC1 regulates the expression of some
B3 genes only locally.
In conclusion, by focusing on At2S3 regulation, we have shown that FUS3 and LEC2 have a similar mode of action that differs from that of ABI3. This finding is consistent with the emerging model of an At2S3 regulatory complex containing several DNA binding proteins (FUS3, LEC2 and bZIPs) and ABI3 as coactivator (Fig. 6B). Many experiments are now possible to test this model biochemically, in yeast, or genetically in planta. Finally, we think that discovering a local regulation between two major players in seed maturation (FUS3 and LEC2) was not only useful for understanding the lec2 phenotype but also suggests the existence of other regulations of this type in the embryo.
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ACKNOWLEDGMENTS |
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Footnotes |
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