1 Department of Molecular Biology, Max Planck Institute for Developmental
Biology, D-72076 Tübingen, Germany
2 Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla,
CA 92037, USA
3 Center for Plant Environmental Stress Physiology, Purdue University, West
Lafayette, IN 47907, USA
Author for correspondence (e-mail:
weigel{at}weigelworld.org)
Accepted 22 August 2003
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SUMMARY |
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Key words: Arabidopsis, Floral induction, Flower development, Floral homeotic genes, Microarrays
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Introduction |
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Flowering is being studied extensively in the reference plant
Arabidopsis thaliana, an ephemeral weed of the crucifer family
(Lohmann and Weigel, 2002;
Simpson and Dean, 2002
). Many
wild Arabidopsis strains flower only after several months unless they
have experienced an extended period of cold, called vernalization. The
vernalization requirement is conferred by a pair of epistatic loci,
FRIGIDA (FRI) and FLOWERING LOCUS C (FLC),
with FLC acting downstream of FRI. In plants with functional
FRI, RNA levels of the floral repressor FLC are high unless
the plants have been vernalized. FLC is also upregulated when genes
of the so-called autonomous pathway are defective
(Michaels and Amasino, 1999
;
Sheldon et al., 1999
).
When FLC is only weakly active, Arabidopsis strains
typically flower within a few weeks under long days, but considerably later
when days are short. The effects of photoperiod variation are mediated by a
signaling cascade that converges on the CONSTANS (CO) transcription factor
(Suárez-López et al.,
2001; Yanovsky and Kay,
2002
), so named because co mutants are much less
responsive to changes in day length than wild-type plants are
(Redeí, 1962
). CO acts
redundantly with a pathway that requires the phytohormone gibberellin, and
gibberellin-deficient co mutants often do not flower at all, even
under long days (Reeves and Coupland,
2001
).
The different floral induction pathways are integrated by a small set of
genes, including FLOWERING LOCUS T (FT), SUPPRESSOR OF
OVEREXPRESSION OF CO 1 (SOC1) and LEAFY (LFY)
(Blázquez and Weigel,
2000; Borner et al.,
2000
; Lee et al.,
2000
; Samach et al.,
2000
). LFY, together with another transcription factor, APETALA1
(AP1), activates homeotic genes such as APETALA3 (AP3) and
AGAMOUS (AG), which specify the identity of the different
organ types in newly arising floral primordia
(Busch et al., 1999
;
Lamb et al., 2002
;
Ng and Yanofsky, 2001
).
The critical events of early flower development are confined to a small part of the plant, the shoot apex, where flowers are initiated. To dissect the interactions between several of the floral regulators on a genome-wide scale, we have used global transcriptional profiling to investigate the response to photoperiod induction at the shoot apex. Our results reveal not only a molecular picture of the interplay between the floral repressor FLC and the photoperiod pathway, but also reveal discrete steps in the acquisition of floral identity. Finally, we identify a large class of genes that are repressed upon floral induction by photoperiod. Potential microRNA targets are found among both the induced and repressed genes.
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Materials and methods |
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Wild type was either Landsberg erecta (Ler) or Columbia
(Col-0, Col-7). In experiments II and III, Ler and Col-7 contained
AG::GUS transgenes (Busch et al.,
1999). lfy-12 is a strong allele in the Col-0 background
(Huala and Sussex, 1992
;
Weigel et al., 1992
) and
co-2 and ft-2 are strong alleles in the Ler
background (Kardailsky et al.,
1999
; Kobayashi et al.,
1999
; Koornneef et al.,
1991
; Putterill et al.,
1995
). The FLC FRI-Sf2 strain contains the FRI
allele of the San Feliu-2 (Sf-2) accession introgressed into Col-0
(Lee et al., 1993
).
flc-3 is a strong loss-of-function allele induced in the FLC
FRI-Sf2 strain (Michaels and Amasino,
1999
).
Scanning electron microcopy (SEM)
After fixation in methanol for 5 minutes, apices were transferred to 100%
ethanol. Further preparation for SEM was as described previously
(Weigel and Glazebrook, 2002).
Images were acquired on a Hitachi S800 electron microscope, at an accelerating
voltage of 20 kV.
RNA isolation and labeling
For RNA isolation from shoot apices, plants were dissected with razor
blades under the dissecting microscope at 30x magnification. Shoot
apices with floral primordia up to about stage 6
(Smyth et al., 1990), or with
equivalently sized leaf primordia, were frozen in liquid nitrogen. Because the
expression of many floral regulators is under circadian control, shoot apices
were harvested starting 1 hour after subjective dawn in about five groups of
five from each genotype, and genotypes were rotated during the collection (it
takes about 1 minute to dissect a shoot apex). Frozen tissue was stored at
80°C, and RNA was extracted with the Plant RNeasy Mini kit
(Qiagen). 5 µg total RNA was used as starting material to synthesize double
stranded cDNA using the Superscript Choice System (Invitrogen) and an
oligo(dT)-T7 primer (Genset). The cDNA served as a template for synthesis of
biotinylated cRNA using the BioArray High Yield Transcript Labeling kit
(Enzo). Biotinylated cRNA was cleaned with RNeasy columns (Qiagen) according
to the manufacturer's protocol, with the following modifications. First, the
cRNA was passed through the column twice to increase binding. Second, the
eluate was re-applied to the column once to increase yield. Usually, 50 to 100
µg biotinylated cRNA were obtained. 20 µg of concentration-adjusted cRNA
were fragmented according to the GeneChip protocol (Affymetrix).
DNA isolation and labeling
Genomic DNA was isolated by a modified CTAB method. 2 g of tissue frozen in
liquid nitrogen was ground up and suspended in 30 ml extraction buffer (0.35 M
sorbitol, 0.1 M Tris pH 8.0, 50 mM EDTA). After centrifugation, the pellet was
resuspended in 2 ml extraction buffer and carefully mixed with 2 ml lysis
buffer (20 mM Tris pH 7.5, 50 mM EDTA, 2 M NaCl, 2% CTAB) and 150 µl
N-laurylsarcosine. Incubation at 65°C for 20 minutes was followed by
extraction with 8 ml chloroform. After precipitation with isopropanol and
sodium acetate, DNA was extracted three times with
phenol:chloroform:isoamylalcohol (25:24:1) and once with chloroform,
precipitated again with ethanol, and resuspended in 100 µl TE buffer. DNA
was fragmented by overnight digestion at 37°C using restriction enzymes
AluI and MseI, followed by heat inactivation of the enzymes
at 65°C for 20 minutes. DNA was extracted with
phenol:chloroform:isoamylalcohol and precipitated with ethanol and sodium
acetate. DNA fragments were labeled using the BioPrime System (Invitrogen)
according to the manufacturer's protocol. Labeled DNA was resuspended in 30
µl nuclease-free water and quantified by spectrophotometry. After DNA
quality was determined by agarose gel electrophoresis, four individual
labeling reactions were pooled to yield at least 30 µg of DNA for
hybridization.
Array hybridization
Hybridization of GeneChip arrays was done according the manufacturer's
protocol (Affymetrix). For washing and staining, protocol EukGe-WS2v4
(Affymetrix) was used. Because there was considerable variation between DNA
hybridization experiments, only arrays hybridized with DNA extracted and
labeled at the same time were compared (two each for Col and Ler).
Using previously described algorithms
(Borevitz et al., 2003), all
unique features were evaluated for differential hybridization. With 3,806
single feature polymorphisms (SFPs) detected among 92,924 unique features, a
false discovery rate of 5.4% was estimated, a number similar to the one
reported before (Borevitz et al.,
2003
).
Analysis of expression data
Expression levels were estimated from Affymetrix hybridization intensity
data using the robust multi array analysis (RMA) package implemented in R
(Irizarry et al., 2003), or
MicroArray Suite 5.0 (Affymetrix,
2001
). Expression values were imported into GeneSpring 5.1
(Silicon Genetics) and normalized to the 50th percentile of each array for
further analysis.
Analysis of DNA hybridization
Scanned images were saved as .CEL files using default settings of
MicroArray Suite 5.0 (Affymetrix). Numeric values representing the signal of
each feature were analyzed using scripts and statistical methods developed by
Borevitz and colleagues (Borevitz et al.,
2003) and implemented in R.
Identification of Col/Ler length polymorphisms
Primers located in the 5' and 3' UTRs of candidate polymorphic
genes are listed in Table S1 at
http://dev.biologists.org/supplemental.
Genomic DNA was purified with the DNeasy Plant Mini kit (Qiagen). PCR was
carried out using a 1:10 mixture of ExTaq (Takara) and Taq polymerase in ExTaq
buffer with 10 pmol of each primer and 50 ng of DNA in 20 µl volume. PCR
reactions were cycled for 41 times at 94°C for 20 seconds, 51°C for 30
seconds and 72°C for 5 minutes.
Real time and semi-quantitative RT-PCR
Total RNA was extracted from apices of plants grown in an independent
experiment using RNeasy Mini columns with on-column DNAse digestion (Qiagen).
Reverse transcription was performed with 1 µg of total RNA, using a Reverse
Transcription Kit (Promega). PCR amplification was carried out in the presence
of the double-strand DNA-specific dye SYBR Green (Molecular Probes).
Amplification was monitored in real time with the Opticon Continuous
Fluorescence Detection System (MJR). A list of primers used is shown in Table
S2
(http://dev.biologists.org/supplemental).
Identification and analysis of the ALF7 mutant
Arabidopsis plants of the Col strain were transformed with the
pSKI015 activation-tagging vector (Weigel
et al., 2000) and several Activation-tagged
Late-Flowering (ALF) lines were selected in the
T1 generation. Plasmid rescue was used to identify the insertion
point in one of these lines, ALF7. The corresponding cDNA and that of its
paralog were PCR-amplified from first-strand cDNA generated from shoot apex
RNA, and placed behind the CaMV 35S promoter in the pART27 derivative pMLBART
(Gleave, 1992
). The resulting
constructs were introduced into Col wild type by Agrobacterium
tumefaciens-mediated transformation
(Weigel and Glazebrook,
2002
).
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Results and discussion |
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|
|
When we used the ATH1 array to compare RNA signals of vegetative shoot apices from Col and Ler, we found 961 transcripts with at least a two-fold difference in signal intensity between the two accessions. 553 of these had a lower signal in Ler, again raising the possibility that some of them might be polymorphic. However, 408 transcripts produced a higher signal in Ler, suggesting that these differences are true expression changes (see Fig. S1 at http://dev.biologists.org/supplemental). In conclusion, sequence polymorphisms or deletions in the Ler sequence should not be a major concern when using Affymetrix arrays for analysis of Ler-derived samples. However, there appear to be many genuine expression differences between Col and Ler (Fig. 1C,D; Fig. S1 at http://dev.biologists.org/supplemental), and it is important to consider this fact when comparing non-isogenic strains.
Effect of day length change on two different wild-type strains
To monitor changes in gene expression during floral induction and early
flower development, we grew plants under short photoperiods (which delays
flowering) for 30 days, and then transferred them to long days. In a pilot
experiment, we had found that many flower-specific markers such as homeotic
genes were not detected on day 0, but were robustly induced around day 6.
Scanning electron microscopy confirmed that the shoot apex was vegetative at
the beginning of the experiment (Fig.
2A,C). After wild-type plants had been grown in long photoperiods
for 7 days, the oldest floral primordia at the end of our experiments were
around stage 7 (Smyth et al.,
1990). Importantly, in addition to floral primordia, release of
lateral shoot primordia was evident
(Hempel and Feldman, 1994
).
Thus, we can expect to identify in our experiments at least three classes of
genes in addition to genes that are expressed in young flowers: genes that
characterize young leaf primordia (which should be repressed); genes that mark
the formation of side shoots (which should be induced), and genes that
distinguish the shoot apical meristem before and after floral induction.
|
Several genes are known to be induced in the shoot meristem proper upon
floral induction, including the MADS box genes SOC1 and
FRUITFULL (FUL), the SQUAMOSA PROMOTER BINDING PROTEIN
LIKE 3 (SPL3), SPL4 and SPL5 genes and the
REM1 gene (Borner et al.,
2000; Cardon et al.,
1999
; Cardon et al.,
1997
; Franco-Zorrilla et al.,
2002
; Hempel et al.,
1997
; Lee et al.,
2000
; Samach et al.,
2000
). Other genes, such as FLOWERING PROMOTING FACTOR1
(FPF1), are induced at the periphery
(Kania et al., 1997
). For the
floral primordia proper (Smyth et al.,
1990
), several stage-specific markers are known. During stage 1,
the floral meristem identity genes LFY, AP1 and the AP1
paralog CAULIFLOWER (CAL) are induced
(Gustafson-Brown et al., 1994
;
Kempin et al., 1995
;
Weigel et al., 1992
). During
stage 2, SEPALLATA1 (SEP1), SEP2 and SEP3
are activated, and shortly thereafter the homeotic genes AP3, PI and
AG, which act in combination with the SEP genes
(Drews et al., 1991
;
Flanagan and Ma, 1994
;
Goto and Meyerowitz, 1994
;
Jack et al., 1992
;
Savidge et al., 1995
).
Upregulation of all genes discussed above was easily detected in both Col and
Ler samples (Fig.
3A-F). For the later time points, the MAS software identified
almost all of them `present', which is an indication of the ease with which
these genes are detected.
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All floral markers were induced more quickly in Ler than in Col.
Both strains are relatively early flowering compared to many wild accessions,
partially because they have null alleles at the FRIGIDA
(FRI) locus, which is required for high expression of the floral
repressor FLC (Johanson et al.,
2000; Michaels and Amasino,
1999
; Sheldon et al.,
1999
). However, Col flowers several leaves later than Ler
under long days (e.g. Kardailsky et al.,
1999
). One genetic difference between the two strains is that the
Ler allele of the floral repressor FLC is only very weakly
active (Koornneef et al.,
1994
; Lee et al.,
1994
). Accordingly, we detected lower FLC levels in
Ler than in Col (Fig. S3,
http://dev.biologists.org/supplemental).
CO- and FT-dependent targets of floral
induction
Activity of the CO gene is essential for perception of photoperiod
differences (Koornneef et al.,
1991). CO acts through at least two other genes with
major effects on flowering time, FT and SOC1
(Samach et al., 2000
;
Suárez-López et al.,
2001
). Loss-of-function mutations in all three genes delay
flowering under long days, with co mutations having the strongest and
soc1 the weakest effects
(Koornneef et al., 1998
;
Onouchi et al., 2000
). Because
FT and SOC1 integrate other cues in addition to photoperiod,
mutations in both genes also delay flowering under short days, where
co mutants are normal (Borner et
al., 2000
; Koornneef et al.,
1991
; Lee et al.,
2000
; Onouchi et al.,
2000
). To assess whether all effects of day length on gene
expression in the shoot apex are transduced by the CO pathway, and
how much of the CO effect is mediated by FT, we compared the
expression profiles of Ler wild-type plants to those of co-2
and ft-2 mutants. By the end of our experiments, floral primordia
were just beginning to form in co-2 and ft-2 mutants
(Fig. 2I,J).
An examination of known floral marker genes revealed that co and
ft had very similar effects (Fig.
3). Overall, the effects of co and ft reflected
the sequence of induction in wild type. That is, early response genes, such as
FUL, SOC1 and SPL3-5, were attenuated, with FUL
showing the smallest change compared to wild type
(Fig. 3A,B). Induction of
LFY was only attenuated (Fig.
3D). Interestingly, FPF1, which is expressed in a similar
temporal pattern as LFY in wild type, is affected more strongly than
LFY by co and ft
(Fig. 3C). There were several
other genes whose expression profile across all data sets was highly
correlated with that of FUL (>90%), including that of the
SOC1 paralog AGL42 (Fig.
3A). The other floral markers, including AP1, CAL, the
SEP genes and the homeotic genes AP3, PI and AG,
were not induced in co or ft during the time course of the
experiment (Fig. 3D-F).
Finally, induction of CRC was only moderately attenuated in
co and ft mutants (Fig. S2,
http://dev.biologists.org/supplemental).
This observation confirms that the CRC expression detected here must
be different from the highly localized expression in carpels
(Bowman and Smyth, 1999), since
neither co nor ft mutants had produced stage 6 flowers by
the end of the experiment.
It is notable that SOC1 was affected not only by co, but
to a similar extent by ft, indicating cross-regulation between the
two CO targets, FT and SOC1. LFY, which is
expressed weakly during the vegetative phase
(Blázquez et al., 1997;
Hempel et al., 1997
), was
identified as `present' by the MAS software prior to floral induction. The
induction of LFY is attenuated in co mutants, but also in
ft mutants, even though genetic analyses clearly show FT and
LFY to act in parallel
(Kardailsky et al., 1999
;
Kobayashi et al., 1999
;
Nilsson et al., 1998
;
Ruiz-García et al.,
1997
).
We used reverse transcription followed by quantitative (real-time) PCR to confirm the expression changes of several of these genes in wild type and mutants, using RNAs prepared in a separate experiment from plants at 0 and 7 days after transfer to long days (Fig. S4, http://dev.biologists.org/supplemental). All genes tested were induced more strongly in wild-type plants than in the corresponding mutants, confirming the effects. RT-qPCR resulted in higher estimates for induction of the early marker genes (FUL, CAL, AP1) in Col than in Ler, which contrasts with the interpretation of the Affymetrix array data. This may either be due to the fact that this was an independent experiment or to differences in amplification efficiencies for Col and Ler samples.
To compare the effects of co and ft more broadly, we selected those genes that changed the most during the time course of the experiment. Using RMA, we calculated for all genes the absolute average difference in expression levels between days 0 and 7 for the replicate Ler and Col sets. We then ranked all genes by expression change and selected the overlap between the top 500 genes in both Ler and Col (`top 500 list'). This cutoff corresponded to a 2.6-fold change in Ler and a 1.9-fold change in Col, which reflects the more dramatic responses seen with known flowering genes in Ler.
This procedure is conservative, since it removes several genes that are detected robustly in only one of the two accessions. Nevertheless, there was a remarkable overlap between the Ler and Col sets. For genes with increased expression, the overlapping 101 genes represented 73% and 54% of the corresponding Ler and Col sets, respectively. For genes with decreased expression, the overlapping 231 genes represented 63% and 74% of the corresponding Ler and Col sets, respectively. A comparison of results for this list of genes from two replicate arrays for individual genotype-time point combinations demonstrated that the signals for most of these genes are readily reproducible (Fig. 4A). The effects of the filter are obvious in a scatter plot comparing Ler (day 0) with Ler (day 7) (Fig. 4B).
|
In addition to FT and SOC1, two other CO
targets, ACS10 and P5CS2, have been found using an inducible
form of CO (Samach et al.,
2000). Both genes were detected at high levels in all genotypes
that were analyzed, but their levels did not change during the course of the
experiment (Fig. S5,
http://dev.biologists.org/supplemental).
A possible explanation for the discrepancy is that we analyzed only material
from the shoot apex, whereas Samach and colleagues
(Samach et al., 2000
) analyzed
whole seedlings.
Integration of photoperiod and FLC activity
FLC is an important repressor of flowering that acts in parallel
with the photoperiod pathway (Borner et
al., 2000; Lee et al.,
2000
; Samach et al.,
2000
). Because of a deletion in the FLC activator
FRI (Johanson et al.,
2000
), FLC levels are much reduced in Col compared to an
isogenic strain with the functional FRI-Sf2 allele (Fig. S3,
http://dev.biologists.org/supplemental)
(Lee et al., 1993
). To
determine the effects of FLC on the acute response to photoperiod
induction, we compared the expression of floral markers in the congenic
strains FLC FRI-Sf2, FLC fri-Col (Col wild type), flc-3
FRI-Sf2 and flc-3 fri-Col
(Michaels and Amasino, 2001
).
Integration of photoperiod and autonomous pathways appears to be downstream of
CO, since CO displays a similar induction profile in all
four genotypes. We found that early induction of CAL was only
moderately attenuated by FLC activity, whereas SOC1
induction was severely affected, but still detectable. In contrast,
FUL induction was abolished in FLC FRI-Sf2
(Fig. 5). Thus, FLC
appears to act additively with some regulators of the photoperiod pathway and
epistatically with others, consistent with the notion that FLC and
CO activities are integrated by the same promoters
(Hepworth et al., 2002
).
|
We found that only a minority of substantial expression changes caused by
transfer from short to long days was LFY dependent. In addition to
known LFY targets, which are the homeotic genes AP1, AP3, PI
and AG (Fig. 3F)
(Busch et al., 1999;
Lamb et al., 2002
;
Liljegren et al., 1999
;
Wagner et al., 1999
;
Weigel and Meyerowitz, 1993
),
the group of LFY-dependent genes includes the homeotic cofactors
SEP1-3 (Fig. 3E); all
7 genes are also found in the `Col and Ler top 500' list. A less
dramatic effect was seen for the AP1 paralog CAL
(Fig. 3D).
Next, we mined the expression profiles for genes that behaved similarly to the homeotic or the SEP genes across all data sets. This procedure resulted in 10 additional genes, of which 6 were again in the `Col and Ler top 500' list (Fig. 6, Table 2). None of them was as strongly induced as the most obvious LFY targets, such as AP1, AP3 or PI. As expected, additional analyses did not identify any genes that were dependent on LFY, but not on CO or FT.
|
|
Zik and Irish (Zik and Irish,
2003) have recently reported an analysis of the response of about
6,000 genes to changes in activity of the LFY targets AP3
and PI. The authors identified 47 potential AP3/PI targets,
of which 42 are represented on the ATH1 array. Among these, we found only one
gene, At5g22430, that is obviously affected in lfy mutants
(Fig. 6B).
Genes repressed upon floral induction
An unanticipated finding was that there are considerably more genes that
are repressed upon transfer from short to long days than are induced; from our
`Col and Ler top 500' list, 101 genes were activated and 231 genes
repressed (Fig. 7; see Table S4
at
http://dev.biologists.org/supplemental,
for a list of genes and their expression values). We do not think this is an
artifact, because we see a similar ratio if we include a wider range, e.g.,
top 1000 genes. Previous molecular screens have focused on genes that are
activated upon floral induction (e.g.
Franco-Zorrilla et al., 1999;
Melzer et al., 1990
;
Samach et al., 2000
).
Similarly, although forward genetic screens have identified several floral
repressors (Mouradov et al.,
2002
; Simpson and Dean,
2002
), only one of them, FLC, is known to be
down-regulated by vernalization, a treatment that promotes flowering
(Michaels and Amasino, 1999
;
Sheldon et al., 1999
), and
none has been identified that is repressed by photoperiod. A more detailed
inspection of a subset of repressed genes showed that their behavior was
opposite to that of the induced genes across all genotypes, i.e., the
Ler response was faster than that of Col, and repression was not
completely absent in co and ft mutants
(Fig. 8).
|
|
A pair of paralogous AP2-domain genes that can repress flowering
An important question is, of course, whether any of the repressed genes
play an instructive role in flowering. Coincidentally, we isolated a dominant,
activation-tagged late-flowering line, ALF7. Plasmid rescue showed that the
activation-tagging vector (Weigel et al.,
2000) was inserted next to gene At3g54990, which encodes an
AP2-domain protein that we named SCHLAFMÜTZE (SMZ)
(Fig. 9A). Analysis of our
expression data showed that this gene was repressed upon photoperiod change in
Col and Ler wild type as well as lfy mutants, but not in
co or ft mutants (Fig.
9B). At3g54990 has a close homolog, At2g39250, which is expressed
at lower levels and which was named SCHNARCHZAPFEN (SNZ).
The expression profiles of SMZ and SNZ were similar when
analyzed by MAS, but down-regulation of SNZ was less apparent when
analyzed by RMA (Fig. 9B). For
both genes, we generated several transformants in which the coding sequences
were placed behind the constitutive 35S promoter from cauliflower
mosaic virus. Several lines in which SMZ or SNZ were under
the control of the 35S promoter flowered much later than wild type
(Fig. 9C), confirming that
SMZ and SNZ can repress flowering. Consistent with redundant
function of the two genes, SNZ knockouts flower normally. Although
SMZ insertions are available, these do not interfere with RNA
expression (data not shown).
|
MiRNA-guided degradation of specific mRNAs has recently been demonstrated
to be important for plant morphogenesis
(Palatnik et al., 2003). For
the four SMZ and SNZ-related genes, Kasschau and colleagues
(Kasschau et al., 2003
) have
shown that at least a fraction of their mRNAs is cleaved in wild-type
inflorescences in the middle of the region that is complementary to the miR172
miRNAs. Experiments with dcl1 mutants and RNA blots indicate that
mRNA cleavage is frequent in RAP2.7 and At5g60120, and rarer for
AP2 and At5g67180 (Kasschau et
al., 2003
). SMZ and SNZ share the miR172
complementary motif, but with 3 or 4 mismatches (Fig. S6,
http://dev.biologists.org/supplemental).
Among the other four, only At5g67180 has also at least 3 mismatches, while the
remaining three have 1 or 2 mismatches with at least one miR172 isoform. When
we examined the expression profiles of this clade of AP2 domain encoding
genes, we found that AP2, RAP2.7 and At5g60120 are
down-regulated similarly to SMZ, and that their down-regulation is
CO and FT dependent. The expression levels of
At5g67180 also responded to floral induction, but in an opposite
manner (Fig. 10A).
|
The three up-regulated SPL genes discussed earlier, SPL3,
SPL4 and SPL5 (Fig.
3B), have also been identified as miRNA targets
(Kasschau et al., 2003;
Rhoades et al., 2002
). When we
examined the other SPL genes represented on the Affymetrix array, we
found that SPL2, SPL6, SPL9, SPL10, SPL11, SPL13 and SPL15
behave similarly to SPL3, SPL4 and SPL5, but that they
reacted less strongly to floral induction
(Fig. 10C). We noted that the
latter three are distinguished from the rest by the presence of the miR156
miRNA target motifs in the 3' UTR rather than the coding sequence.
![]() |
Conclusions |
---|
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---|
The parallel analysis of many known floral regulatory genes, along with the
analysis of a large group of newly identified genes that respond to a change
in photoperiod, has allowed us to draw several important conclusions. First,
two genes previously identified as CO targets by CO
overexpression, ACS10 and PC5S2, do not change at the shoot
apex, implying that CO also affects processes outside the region
where flowers are formed. Second, consistent with the observation that among
the two other known CO targets, FT has more dramatic effects
than SOC1 (Onouchi et al.,
2000; Samach et al.,
2000
), the very similar expression profiles of co and
ft mutants suggest that, at the shoot apex, FT is the major
output of CO. Third, the effects of the floral repressor FLC
and photoperiod are additive, resulting in expression profiles of floral
marker genes that are similar in plants with and without FLC, but
with overall much lower levels in the presence of FLC. This finding
also confirms that the similar expression profiles of co and
ft are not simply due to the fact that flower formation is delayed in
both mutants, since plants with high FLC levels flower even later
than co or ft mutants. Fourth, compared to CO and
FT, a mutation in LFY has much more subtle effects,
indicating that LFY acts further downstream in the floral induction
cascade, even though genetically FT and LFY act in parallel
downstream of CO.
There are several additional discoveries that we have made by inspecting our data set for genes without a known role in flowering. First, we found that forward genetic analysis has been very successful in identifying many of the genes that are most strongly activated in response to floral induction. However, an equally important response to floral induction may be the repression of regulatory genes. That at least some of these repressed genes indeed have a role in flowering is confirmed by the analysis of the SMZ and SNZ genes. Second, two classes of transcription factor genes, one coding for MADS domain proteins and the other for SBP domain proteins, are highly overrepresented among the genes that are induced in response to photoperiod, both when compared to the overall complement of these families in the genome and when compared to the class of repressed genes. This observation suggests that flower-specific expression is the ancestral state for many genes in these two families. We have also found that there is a large class of genes that produce differential RNA signals between two different wild-type strains, Col and Ler (Fig. S1, http://dev.biologists.org/supplemental), which provides a rich source of candidates controlling phenotypic differences between these two strains.
How floral inductive signals are transmitted from genes such as CO
and FT to downstream effectors such as LFY and AP1
is not well understood, and the newly discovered set of genes dependent on
CO and FT, but not LFY, constitute a source of
potential factors playing important roles in this process. We noticed several
paralogous gene pairs with very similar CO and FT responses
in this group, which suggests that many of these genes were not identified in
forward genetic screens because of redundancy. We have discovered two groups
of potential miRNA targets, a clade of AP2-domain-encoding genes and a large
group of SPL genes, as being regulated by CO and
FT. This observation raises the possibility that miRNAs perform a
critical function in mediating the effects of floral induction, which is
supported by a recent report on the consequences of miR172 overexpression
(Chen, 2003). The analysis of
other flowering mutants in a similar experimental design as the one used here
should further clarify the regulatory interactions between the many genes
already known to play a role in flowering.
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ACKNOWLEDGMENTS |
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Footnotes |
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Aukerman and Sakai recently showed that At2g28550 (named TOE1) and
At5g60120 (named TOE2) are also floral repressors
(Aukerman and Sakai, 2003).
* Present address: German Cancer Research Center, 69120 Heidelberg,
Germany
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