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Regulation of FLOWERING LOCUS T by a MicroRNA in
Brachypodium distachyon
CW
Liang Wu,a,1 Dongfeng Liu,a,b,c ,1 Jiajie Wu,d Rongzhi Zhang,a Zhengrui Qin,a Danmei Liu,a Aili Li,a Daolin Fu,d
Wenxue Zhai,b,c and Long Maoa,2
a National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of
Agricultural Sciences, Beijing 100081, China
b Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing100101, China
c University of Chinese Academy of Sciences, Beijing 100049, China
d State Key Laboratory of Crop Biology, Shandong Agricultural University, Taian 271000, China
ORCID IDs: 0000-0003-4953-7258 (L.W.); 0000-0002-3377-4040 (L.M.).
The highly conserved florigen gene FLOWERING LOCUS T (FT) functions at the core of the flowering pathways. Extensive
studies have examined the transcriptional regulation of FT; however, other layers of FT regulation remain unclear. Here, we
identified miR2032, a Pooideae-specific microRNA that is expressed in leaves and targets Brachypodium distachyon FT
orthologs for mRNA cleavage. miR2032 was abundantly expressed in plants grown under short-day (SD) conditions but was
dramatically repressed in plants transferred to long-day (LD) conditions. We also found that the epigenetic chromatin status,
specifically the levels of histone methylation marks, at miR2032 precursor loci changed in response to daylength. Moreover,
artificial interruption of miR2032 activity by target mimicry in B. distachyon altered flowering time in SD but not in LD
conditions, suggesting that miR2032 functions in photoperiod-mediated flowering time regulation. Together, these findings
illustrate a posttranscriptional regulation mechanism of FT and provide insights into understanding of the multiple concerted
pathways for flowering time control in plants.
INTRODUCTION
The transition from vegetative growth to flowering stage is
a critical event for seed propagation in plants. The correct timing
of flowering initiation depends on five genetic pathways: photoperiod, vernalization, autonomy, hormones, and age (Fornara
et al., 2010). These endogenous and environmental flowering
regulatory cues ultimately regulate a set of integrator genes,
such as SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1
(SOC1), LEAFY, and FLOWERING LOCUS T (FT) (Kobayashi and
Weigel, 2007).
Photoperiodic control of flowering in plants begins with the
perception of daylength in leaves, followed by the transmission
of a floral signal termed florigen into the shoot apex where the
flowers are initiated (Bäurle and Dean, 2006). The mobile florigen
signal is encoded by FT and FT orthologous genes in various
plant species (Lifschitz et al., 2006; Tamaki et al., 2007; Pin
et al., 2010; Meng et al., 2011; Navarro et al., 2011; Wigge,
2011). Mutation in FT causes a serious delay in flowering, whereas
overexpression of FT induces precocious flowering, indicating
that FT is necessary and sufficient to accelerate the floral transition
1 These
authors contributed equally to this work.
correspondence to [email protected].
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) are: Liang Wu
([email protected]) and Long Mao ([email protected]).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.118620
2 Address
(Kardailsky et al., 1999; Kobayashi et al., 1999). In Arabidopsis
thaliana, FT protein travels from the vasculature to the shoot
apex to form an effector complex with FLOWERING LOCUS D,
a bZIP transcription factor, and thereby stimulates flower organ
formation through activation of a subset of downstream floral
genes, including APETALA1 (AP1), FRUITFULL (FUL), and SOC1
(Abe et al., 2005; Wigge et al., 2005; Corbesier et al., 2007; Mathieu
et al., 2007).
Positive and negative regulators control FT transcription. Under
long-day (LD) conditions, FT expression is mainly activated in
vascular bundles via transcriptional regulators CONSTANS (CO)
and GIGANTEA (Suárez-López et al., 2001; Sawa and Kay, 2011;
Torti et al., 2012). CO is directly recruited to the FT promoter or
indirectly acts with other transcriptional cofactors to regulate FT
expression (Wenkel et al., 2006; Kobayashi and Weigel, 2007).
The age-dependent SQUAMOSA PROMOTER BINDING PROTEINLIKE3 can also directly act at the FT promoter and modulate
ambient temperature-responsive flowering (Wang et al., 2009; Wu
et al., 2009a; Kim et al., 2012). As a major negative regulator of FT,
FLOWERING LOCUS C forms a MADS box complex with SHORT
VEGETATIVE PHASE, which directly inhibits FT expression to
mediate the effects of exposure to cold conditions or ambient
temperature in Arabidopsis (Bastow et al., 2004; Bäurle and Dean,
2006; Li et al., 2008). Furthermore, two TEMPRANILLO proteins
(TEM1 and TEM2) directly repress FT and may have antagonistic
roles with the activator CO to determine FT levels (Castillejo and
Pelaz, 2008). Several miR172-targeted AP2-like genes also affect
the repression of FT transcription, but their modes of action remain unclear (Mathieu et al., 2009).
In addition to transcription factors, trimethylation of H3K27
(H3K27me3) and demethylation of H3K4 (H3K4me2) modifications
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
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in cis also epigenetically regulate FT transcription (Farrona et al.,
2008; Adrian et al., 2010). FT might also be controlled at posttranscriptional levels, since the werewolf root hair patterning
mutant indirectly affected FT mRNA stability in Arabidopsis, but
the molecular basis of this process is still elusive (Seo et al.,
2011).
Compared with eudicots, the behaviors of FT during floral
induction in monocots are largely unknown to date. In rice (Oryza
sativa), a typical short-day (SD) plant, two FT-like proteins referred to as Heading-date3a and RICE FLOWERING LOCUS T1,
which are respectively activated under SD and LD conditions,
have been proposed to be the SD- and LD-specific florigens
(Komiya et al., 2009). Ectopic expression of the FT-like gene
VERNALIZATION3 (VRN3) in wheat (Triticum aestivum) and
barley (Hordeum vulgare) activated transcription of the AP1 orthologous gene VRN1 and promoted early flowering (Trevaskis
et al., 2003; Yan et al., 2006).
With its rapid growth, small genome, and simple growth conditions, Brachypodium distachyon has been thought as a suitable
model system for studies on temperate cereals and biofuel plants,
such as switchgrass (Panicum virgatum; Opanowicz et al., 2008);
however, so far, information about its flowering is quite limited
(Higgins et al., 2010; Hong et al., 2010). B. distachyon accessions
show substantial natural variation in flowering time. For example,
the genome-sequenced accession Bd21 is a spring annual and
can flower without any vernalization, whereas the winter accession Bd1-1 requires vernalization, a period of cold treatment, for
flowering (Schwartz et al., 2010). In addition, daylength greatly
influences the flowering time of B. distachyon accessions. For
instance, Bd21 can flower within 45 d of growth under LD conditions but will delay flowering for half a year if grown in SD environments (Schwartz et al., 2010; see Supplemental Figures 1A
and 1B online). Thus, there may be special components that
precisely regulate flowering time in B. distachyon.
The 21- to 24-nucleotide microRNAs (miRNAs), a class of
DICER-LIKE (DCL)–dependent small RNAs (sRNAs), regulate
a wide range of biological processes in monocots and eudicots
(Voinnet, 2009; Wu et al., 2009b). In this study, we characterized
a Pooideae-specific miR2032 that guides sequence-specific
cleavage of FT orthologous transcripts in B. distachyon leaves.
We further demonstrate that miR2032 plays an important role in
regulation of the photoperiod-mediated floral transition. Our
findings show that a miRNA directly mediates FT posttranscriptional modulation in Pooideae plants, which may also
supply an alternative approach for heading date breeding in barley
and wheat.
RESULTS
Hairpin Structures and Primary Transcripts of MiR2032
We previously identified a subset of miRNAs by deep sequencing of
small RNAs in bread wheat. One 21-nucleotide miRNA, miR2032,
was identified, and its targets were predicted in silico to be FT
orthologous genes (Wei et al., 2009). Owing to the importance of
FT-like proteins in plant flowering pathways, we further analyzed
this miRNA in depth.
To determine the conservation of miR2032 in other plant
species, we performed a BLAST search of all available small
RNA databases. We only detected the 21-nucleotide miR2032
sequence in the sRNA data sets of a number of Pooideae
species, including barley and B. distachyon (Schreiber et al.,
2011; Hackenberg et al., 2012). RNA gel blot analysis validated
that miR2032 was present in the three Pooideae plants, but not
in rice (Oryza sativa), maize (Zea mays), and sorghum (Sorghum
bicolor), nor in dicotyledonous plants, such as Arabidopsis
thaliana (Figure 1A). These results suggest that miR2032 is
a recently evolved and Pooideae-specific miRNA (see
Supplemental Figures 2A and 2B online).
Since B. distachyon has emerged as a research model for
Pooideae plants, by virtue of its published high-quality genome
sequence and tractable genetic transformation systems (Vogel
et al., 2010; Brkljacic et al., 2011), we investigated the structure and functions of miR2032 in detail in B. distachyon. Bioinformatic prediction of miR2032 stem-loop structures found
that two loci on B. distachyon chromosome 1 potentially encoded
the miR2032 transcribed genes (Figure 1B). We named these two
miR2032 genes MIR2032a and MIR2032b. Another miRNA, which
was previously annotated as miR5200 in B. distachyon (Zhang
et al., 2009), has only one nucleotide difference with miR2032a
and miR2032b (Figure 1B); we thereby termed this miRNA
miR2032c. Interestingly, like in wheat, the B. distachyon MIR2032c
was located at the same type of miniature inverted terminal repeat
element as MIR2032b, suggesting that both mature sequences
and precursor structures of miR2032 are conserved between
B. distachyon and wheat (Abrouk et al., 2012; Lucas and Budak,
2012).
To better understand the complete gene structures of primary
miR2032 (pri-miR2032), we used RNA ligase-mediated rapid
amplification of cDNA ends (RACE) to retrieve 59 and 39 sequences of the transcript. Like canonical miRNAs (Xie et al.,
2005), we found that both MIR2032a and MIR2032b transcripts
were 59 capped with 39 polyadenylation (see Supplemental
Figures 3A and 3B online). Intriguingly, we detected 14 splice
variants of MIR2032a, but each variant had the same 59 segment
(see Supplemental Figure 3A online). Distinct from various
MIR2032a transcripts that carried one to several introns, the
short MIR2032b transcript had only one exon (see Supplemental
Figure 3B online).
MiR2032 Targets FT Orthologous Genes and Accumulates
in Leaves
Similar to the initial computational prediction of miR2032 targets
in wheat and barley (see Supplemental Figures 4A and 4B online),
using psROBOT sRNA analysis toolbox (Wu et al., 2012), we
identified two B. distachyon FT-like genes (Bradi2g07070 and
Bradi1g48830), which were previously annotated as FTL1 and
FTL2 (Higgins et al., 2010), as miR2032 targets (Figure 1C; see
Supplemental Figures 4C and 4D online). Further validation
through 59 RNA ligase-mediated RACE showed that both FTL1
and FTL2 mRNAs could be cleaved at miR2032 target sites in
vivo, suggesting that these two FTs are bona fide miR2032
target genes (Figure 1C). Although a majority of the cleavage
events of FTL1 occurred outside of the canonical miRNA
Regulation of FLOWERING LOCUS T by a MicroRNA
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Figure 1. Hairpin Structures, Expression Patterns, and Target Validation of MiR2032.
(A) RNA gel blot analysis of miR2032 in wheat, barley, B. distachyon, rice, maize, sorghum, and Arabidopsis. Note that miR444 is a monocot-specific
miRNA, whereas miR172 and miR159 are conserved between monocots and dicots. U6 was used as RNA loading control. nt, nucleotides.
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cleavage site, it is unlikely that this cleavage was mediated by
secondary siRNAs because miR2032 is not 22 nucleotides in
length and there are no bulged bases between the miRNA:
miRNA* (for miRNA star) duplex that are known to be required
for secondary siRNA formation (Chen et al., 2010; Cuperus et al.,
2010; Manavella et al., 2012).
Since FT is not expressed at the shoot apex, it is reasonable to
ask whether miR2032 is also specifically expressed outside the
shoot apex to degrade FT mRNAs. To address this question, we
collected B. distachyon leaves and shoot apices and performed RTPCR to determine miR2032 expression patterns in these tissues
(Figure 1D). Strikingly, we found that both MIR2032a and MIR2032b
dramatically accumulated in leaves, but neither of them appeared in
shoot apex (Figures 1E and 1F). By contrast, AP1 ortholog VRN1
and GAPDH genes were present in both tissues (Figures 1E and 1F).
Together with the observation that DCL1 was expressed in both
leaves and shoot apices, these results suggest that miR2032 is
specifically expressed in leaves to repress FT mRNAs.
To verify that miR2032 targets FTs in vivo, we generated
miR2032 overexpression (miR2032-OE) transgenic B. distachyon
plants to trace whether FTLs were inhibited when miR2032 increased. We obtained 11 independent transgenic lines from
separately generated calluses and all of them showed delayed
flowering time phenotypes in LD environments (see Supplemental
Figures 5A and 5B online). Based upon the severity of late flowering time, these lines were divided into Types I and II (Figure 2A):
Type I did not flower even after 180 d and thus could not form
seeds; Type II bolted much later than the wild type (110 versus 45
d) and produced fewer seeds than the wild-type plants (Figures
2A and 2C). Moreover, both types of miR2032-OE plants eventually grew much higher than the wild type (Figures 2B and 2C).
RT-PCR showed that the expression of FTL1 and FTL2 was
dramatically reduced in Types I and II transgenic lines, whereas
that of another FT-like gene (FTL10) without the miR2032 target
site was comparable to the wild type (Figures 2D and 2E). We also
found that B. distachyon AP1 and FUL orthologous genes,
VRN1 and FUL2, which may act downstream of FT (Li and
Dubcovsky, 2008), also displayed decreased mRNA levels in
miR2032-OE plants (Figures 2D and 2E). These results indicate
that miR2032 mediates FTL1 and FTL2 mRNA degradation for
posttranscriptional regulation.
Photoperiod Affects MiR2032 Expression
The initiation of flowering in temperate grasses depends on delicate regulation of FT, which plays a central role in balancing
competing signals from vernalization and photoperiod (Distelfeld
et al., 2009). To determine whether miR2032 was integrated in FT
regulation in B. distachyon flowering, we examined the expression
patterns of miR2032 in response to vernalization and daylength.
Treating B. distachyon accession Bd21 under 4°C low temperature for 2 to 4 weeks can accelerate plant flowering, but we
observed that mature miR2032 did not change in 2- or 4-week
cold-treated Bd21 plants (see Supplemental Figure 6A online),
suggesting that vernalization may not affect miR2032 in Bd21.
Bd1-1 is a Turkish winter-habit B. distachyon accession and
cannot flower without vernalization (Filiz et al., 2009). However,
2 to 4 weeks of cold treatment did not alter the abundance of
miR2032 in Bd1-1 either (see Supplemental Figure 6A online).
These results reveal that the regulation of FT by miR2032 is
likely to be independent of the vernalization pathway in
B. distachyon.
Next, we examined the amounts of miR2032 in plants grown
under different daylengths. As shown in Figure 3A, RNA gel blots
showed that miR2032 accumulated to high levels in 8-h-light SD
conditions but was nearly undetectable in 16-h-light LD conditions. RT-PCR and quantitative real-time PCR (qPCR) analyses
also revealed that the primary transcripts of MIR2032a and
MIR2032b were much higher in SDs than in LDs (Figures 3A to
3C). These results suggest that miR2032 may be controlled by the
photoperiod. To further assess the reliance of miR2032 on daylength, we detected the abundance of miR2032 under 4-, 8-, 16-,
and 20-h-light conditions. We found that miR2032 had similar
levels under 4- and 8-h-light SD conditions, much higher than in
16- and 20-h-light LDs (see Supplemental Figure 6B online).
Considering that FT family genes usually showed diurnal expression rhythms, we examined the oscillation of pri-miR2032
and targets every 4 h during the day-night cycle in B. distachyon.
Under SDs, both MIR2032a and MIR2032b displayed an expression peak at 4 h before dawn (Figure 3D), and FTLs also
peaked at this time but in LDs (Figure 3E). Furthermore, as
shown in Figures 3D and 3E, MIR2032a and MIR2032b were
expressed at much higher levels, whereas FTL1 and FTL2 accumulated to much lower levels at any time point in SDs than
LDs during the 48-h time-course examination.
In Arabidopsis, FT mRNA levels gradually increased with
growth time, especially under LD conditions (Kobayashi et al.,
1999). Therefore, we asked whether the abundance of miR2032
changed progressively with FT mRNA increases under SDs and
LDs in B. distachyon. Under SDs, FTL1 and FTL2 expression
levels remained constant, whereas miR2032 showed a clear
increase along with growth time (Figures 3F and 3G). Under LDs,
Figure 1. (continued).
(B) The 59 to 39 secondary structures of pre-miR2032 predicted for B. distachyon. The mature miR2032 and miR2032* sequences are indicated in red
and blue, respectively. The different nucleotides between miR2032a/b and c are highlighted by bold letters.
(C) Schematic representation of gene structure and validation of miR2032 targets. Gene exons, introns, and target regions are shown by rectangles,
lines, and green bars, respectively. Arrows indicate the cleavage sites detected by 59-RACE.
(D) Parts of leaf and shoot apex in B. distachyon that were dissected for miR2032 spatial expression analysis.
(E) Spatial expressions of miR2032 and target genes in leaf and shoot apex. The expression of pri-miR2032 and indicated genes was analyzed by
RT-PCR with amplification of GAPDH mRNA as a control.
(F) qPCR analysis of pri-miR2032 and indicated gene expression in leaf and shoot apex. GAPDH was used as an internal control for normalization of
qPCR results. Each point represents the average of three biological replicates, and error bars indicate SD.
Regulation of FLOWERING LOCUS T by a MicroRNA
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Figure 2. MiR2032 Overexpression Severely Delayed Flowering Time in Transgenic B. distachyon Plants.
(A) Flowering time of wild-type (WT) Bd21 and the indicated two types of miR2032-OE transgenic T0 plants. Three plants for type I and eight plants for
type II were scored. Error bars indicate SD.
(B) Plant heights of the wild type and two types of miR2032-OE transgenic T0 plants. Three type I plants and eight type II plants were measured. Error
bars indicate SD.
(C) Phenotypes of ectopic miR2032 expression T0 transgenic B. distachyon plants in LDs. The left plant is 2-month-old wild type. The middle and right
plants are representatives of 4-month-old miR2032-OE transgenic plants with strong and weak phenotypes respectively. White arrows point to spikes.
(D) MiR2032 (by RNA gel-blots) and flowering-time genes (by RT-PCR) expression in wild-type and the indicated miR2032-OE transgenic plants.
Numbers below each miRNA gel blot denote fold changes relative to the miRNA level in wild-type plants.
(E) qPCR analysis of pri-miR2032 and indicated flowering gene expressions in wild-type and the indicated miR2032-OE transgenic plants. GAPDH was
used as an internal control for normalization of qPCR results. Each point represents the average of three technical replicates, and error bars indicate SD.
[See online article for color version of this figure.]
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Figure 3. Photoperiod Affects MiR2032 Expression.
(A) Mature miR2032 (by RNA gel blots), pri-miR2032, and flowering-time genes (by RT-PCR) expression under SDs and LDs. The numbers below each
miRNA gel blot denote fold changes relative to the miRNA level under SDs.
(B) qPCR analysis of pri-miR2032 in B. distachyon plants under SDs and LDs.
(C) qPCR analysis of the indicated flowering gene expressions in SDs and LDs.
(D) Diurnal expression patterns of MIR2032a and MIR2032b in B. distachyon under SDs and LDs.
(E) Diurnal expression patterns of FTL1 and FTL2 under SDs and LDs. Each point represents the average of three technical replicates, and the error bars
indicate repeat SD. The white and black bars along the horizontal axes represent light and dark periods, respectively. The numbers below the horizontal
axes indicate the time in hours.
(F) and (G) Growth stage–dependent expressions of miR2032 (F) and its target genes (G) in SDs. Numbers below each miRNA gel blot indicate fold
changes relative to the miRNA level in 1-week SD plants. w, weeks.
(H) and (I) Growth stage–dependent abundance of miR2032 (H) and its target gene expression (I) under LDs. Each point represents the average of
three biological replicates, and error bar indicates SD. GAPDH was used as an internal control for normalization of qPCR results, and U6 was served
as loading control for RNA gel blots. Numbers below each miRNA gel blot indicate the fold changes relative to the miRNA level in 1-week-old plants
under LDs.
Regulation of FLOWERING LOCUS T by a MicroRNA
both FTL1 and FTL2 showed a gradual increase in expression
along with the photoperiod time; however, we did not detect
miR2032 expression even after 7 weeks (Figures 3H and 3I).
These results suggest that miR2032 appears to specifically inhibit FTL1 and FTL2 accumulation in SDs.
High expression in SDs and low abundance of miR2032 in
LDs as described above implied that photoperiod may affect FT
expression through regulation of miR2032. To further pursue the
effects of daylength on miR2032 and its targets, we examined
the dynamic changes of miR2032 by RNA gel blots, shifting
plants from LDs to SDs and vice versa. When LD-grown plants
were moved from LDs to SDs for 1 d, both pri-miR2032 and
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mature miR2032 were elevated and gradually accumulated to
a high level in 7 d (Figures 4A and 4B). In the meantime, FTL1
progressively decreased to a low level during this period (Figure
4C), suggesting a negative correlation between FTL1 and
miR2032 in this process. When these plants were returned to
SDs for another 7 d, FTL1 and FTL2 displayed a moderate increase along with a reduction of miR2032 (see Supplemental
Figures 6C and 6D online). By contrast, the abundance of mature miR2032 in SD-grown plants remained constant after
transfer to LDs for 7 d even as the pri-miRNA expression began
to decline within 3 d (Figures 4D and 4E). When this shift time
was extended to over 14 d, a gradual reduction of miR2032 was
Figure 4. Dynamic Effects of Daylength Changes on MiR2032, FTL1, and FTL2 Accumulation.
(A) Time course of miR2032 expression when B. distachyon plants were shifted from LDs to SDs. L3w and L4w indicate that B. distachyon were grown
under LDs for 3 and 4 weeks. L1S, L3S, and L7S represent 3-week-old plants that were moved from LDs to SDs for 1, 3, and 7 d, respectively. Numbers
below each miRNA gel blot indicate fold changes relative to the miRNA level in wild-type Bd21 plants grown under LDs for 3 weeks.
(B) qPCR examination of dynamic changes of MIR2032a and MIR2032b in (A).
(C) qPCR analysis of dynamic effects of daylength on FTL1 and FTL2 in (A).
(D) Time course of miR2032 expression when 3-week-old SD plants were transferred to LDs for 1, 3, 7, 14, and 21 d. Plants grown under SDs for 5 and
6 weeks were harvested and used as controls. Numbers below each miRNA gel blot denote fold changes relative to the miRNA level in Bd21 grown
under SDs for 3 weeks.
(E) qPCR examination of dynamic changes of MIR2032a and MIR2032b in (D).
(F) qPCR analysis of dynamic effects of daylength on FTL1 and FTL2 in (D). Error bar means SD (n = 3). Samples for RNA extraction in the experiment
were collected at Zeitgeber time 2. U6 was used as loading control for RNA gel blots. GAPDH was used as an internal control for normalization of qPCR
data.
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Figure 5. Histone Modification Status at Pri-miR2032 Loci in B. distachyon Grown under Different Daylengths.
(A) and (B) Diagrams showing the genomic locations, the adjacent coding genes, and the regions examined by ChIP assay for MIR2032a (A) and
MIR2032b (B). Gray boxes represent exons, and white boxes represent introns. Blue boxes located in gray boxes indicate predicted MIR2032a and
MIR2032b hairpin structures. Positions of mature miR2032 are set as black bars. ChIP regions are shown by black lines.
(C) and (D) Relative abundance of H3K4me3, H3K27me3, and H3K9ac at MIR2032a (C) and MIR2032b (D) genomic regions in B. distachyon grown
under SDs and LDs. Note that the high levels of H3K4me3 and the decreased H3K27me3 around regions corresponding to MIR2032a (P4/P5/P6) and
MIR2032b (P6/P7) hairpin structures are associated with the activation of MIR2032a and MIR2032b expression under SD conditions.
detected (Figure 4D). Interestingly, when these shifted plants
were transferred back to SD conditions for another 7 d, miR2032
expression was recovered (see Supplemental Figure 6E online).
Meanwhile, FTL1 and FTL2 displayed roughly complementary
alterations during these shifts (Figure 4F; see Supplemental
Figure 6F online), suggesting that these two FTLs are photoperiodically regulated by both miR2032 and additional flowering
factors in response to daylength variation.
Changes of Chromatin Structures at Pri-miR2032 Loci
Result in Photoperiodic Expression of miR2032
Epigenetic mechanisms regulate numerous genes in flowering
pathways, including Polycomb repressive complex inhibition of
FT transcription (Farrona et al., 2008). To explore whether the
transcriptional regulation of miR2032 by photoperiod was the
result of epigenetic control, we first examined the status of DNA
methylation at pri-miR2032 loci under SD and LD conditions by
Chop-PCR (see Supplemental Figures 7A and 7B online). We
observed no obvious differences of DNA methylation at
MIR2032a and MIR2032b when B. distachyon was grown in SDs
and LDs (see Supplemental Figures 7C and 7E online). Bisulfite
sequencing of three regions confirmed these results (see
Supplemental Figures 7D and 7F online), suggesting that the
distinct patterns of MIR2032 transcription under different light
conditions are not triggered by DNA methylation.
We then tested histone epigenetic status at MIR2032 loci
under SDs and LDs using chromatin immunoprecipitation (ChIP)
Regulation of FLOWERING LOCUS T by a MicroRNA
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Figure 6. Disruption of MiR2032 Activity in B. distachyon Promotes Flowering under SDs.
(A) A diagram of MIM2032 showing the designed sequences for miR2032 target mimics. UBI, maize ubiquitin promoter; NOS, nos terminator.
(B) Representative photograph of flowering phenotypes in 6-month-old wild-type (WT) and indicated T1 MIM2032 transgenic plants grown in SDs. The
white arrows point to spikes.
(C) Flowering time of wild-type and the indicated T1 MIM2032 transgenic lines grown in SDs. Error bar shows SD (n = 8). Asterisks indicate a significant
difference between wild-type and transgenic plants (Student’s t test, P value < 0.05).
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assay. We designed a series of oligos at both MIR2032 genes
and their neighboring regions (Figures 5A and 5B). Although the
enrichment of positive histone mark H3K9ac around MIR2032a and
MIR2032b loci was similar under SDs and LDs (Figures 5C and 5D),
we found that the repressive histone mark H3K27me3 surrounding
the predicted hairpin structure regions of MIR2032 genes was
enriched much higher under LDs than that under SDs (Figures 5C
and 5D), suggesting that the suppression of pri-miR2032 transcripts under LDs is caused by the high level of H3K27me3 enrichment. H3K4me3 has been reported to act as bivalent mark
together with H3K27me3 at the FT loci in Arabidopsis (Adrian et al.,
2010). Our ChIP data showed that compared with those grown in
LDs, H3K4me3 around the MIR2032a and MIR2032b stem-loop
area was much enhanced when B. distachyon plants were grown
under SDs (Figures 5C and 5D). Furthermore, we found that
H3K4me3 was also highly enriched in the region of MIR2032b 1-kb
terminator under SDs, despite that similar H3K27me3 status was
found at this site in both SDs and LDs (Figure 5D).
Taken together, our results indicate that MIR2032a and MIR2032b
genomic regions are subject to changes in histone modification
under different daylengths. These results suggest that the distinct
expression of miR2032 for photoperiodic regulation of flowering
under SDs and LDs is probably caused by epigenetic chromatin
alterations at MIR2032 loci.
Disruption of MiR2032 Activity Specifically Alters Flowering
Time in SDs
MiR2032 functions in photoperiodic modulation of FTs; therefore, one interesting question is what the consequences are if
the miR2032 regulatory activity is compromised. To address this
issue, we first attempted to generate transgenic lines that ectopically expressed FTL1 and FTL2 as well as their miRNAresistant mutant forms FTL1m and FTL2m, which were resistant
to interaction with miR2032 (see Supplemental Figures 8A, 8B,
and 8D online). Unfortunately, we were unable to generate such
transgenic plants, as all of them flowered extremely early before
they could form roots in the regeneration medium (see
Supplemental Figures 8C and 8E online). These results indicate
that both FTL1 and FTL2 have florigen activity in B. distachyon.
Artificial miRNA target mimics, RNA fragments that have
similar sequence as the miRNA target but cannot be cleaved,
can deplete miRNAs and thus reduce their function, providing
useful reverse genetic tools to investigate biological functions of
individual miRNA families in diverse plants (Franco-Zorrilla et al.,
2007; Todesco et al., 2010; Bergonzi et al., 2013; Zhou et al.,
2013). To gain more insights into photoperiodic regulation of
flowering by miR2032, we designed and generated artificial
target mimics for the miR2032 family and then expressed them
in B. distachyon (Figure 6A). We termed these target mimic
transgenic plants as MIM2032.
As miR2032 was expressed much higher under SDs than LDs,
we first examined the flowering dates of MIM2032 plants in SDs.
We observed that six out of nine independent MIM2032 T1
positive lines flowered 10 to 30 d earlier than wild-type plants
when they were grown in SDs (Figures 6B and 6C), whereas the
T1 segregated negative plants did not show any early heading
phenotypes compared with the wild-type plants (see
Supplemental Figures 9A and 9B online). To illustrate that the
phenotypes of MIM2032 plants in SDs were indeed caused by
the attenuated miR2032 activities, three independent lines were
subject to further molecular analysis. As shown in Figures 6D
and 6E, the expression of miR2032 was reduced in MIM2032
plants, while FTL1 and FTL2 as well as their downstream gene
VRN1 increased. Moreover, we detected the diurnal expression
patterns of FTL1 and FTL2 at 4-h intervals under SDs and found
they exhibited significantly higher expression in MIM2032 than
wild-type plants (Figures 6F and 6G). These findings strongly
suggest that these two FT genes in B. distachyon are indeed
regulated by miR2032 to make plants flower at the correct time
in SDs.
To determine whether disruption of miR2032 activities in
B. distachyon can affect FTs under LD conditions, we extracted
total RNA from LD-grown wild-type and MIM2032 plants and
measured the transcripts of FTLs. We did not detect obvious
differences in expressions of FTL1 and FTL2 or VRN1, indicating
that interruption of miR2032 activities in LDs may not affect FTs
(see Supplemental Figures 9C and 9D online). Consistent with
these results, the diurnal expression of FTL1 and FTL2 was similar
in MIM2032 and wild-type plants under LDs (see Supplemental
Figures 9E and 9F online). Furthermore, we observed that heading
dates were nearly the same for wild-type and MIM2032 plants
under LD environments (see Supplemental Figures 9G and 9H
online). These results imply that miR2032 has little effect on
regulation of B. distachyon flowering time in LD conditions.
Taken together, specific alteration of bolting time of MIM2032
plants in SDs suggests that miR2032 plays an important role in
photoperiod-mediated flowering time regulation.
Photoperiodic Regulation of MiR2032 Appears to Be
Prevalent in the Pooideae
Considering the roles of miR2032 in B. distachyon flowering
control stated above, we asked whether the photoperiodic
Figure 6. (continued).
(D) Expression of miR2032 (by RNA gel blots), targets, and other flowering genes (by RT-PCR) as well as Bradi1g13680 (miR169 target gene as
a control) in wild-type and the indicated T1 MIM2032 transgenic lines under SDs. U6 and GAPDH were served as the RNA gel blot and RT-PCR RNA
loading controls, respectively. The numbers below each miRNA gel blot denote the fold changes relative to the miRNA level in the wild type.
(E) qPCR analysis of miR2032 target genes, VRN1, and Bradi1g13680 (miR169 target as a control) expression in wild-type and the indicated MIM2032
transgenic plants under SDs.
(F) and (G) Diurnal expression patterns of FTL1 (F) and FTL2 (G) in wild-type and the indicated MIM2032 transgenic plants under SDs. Each point
represents the average of three technique replicates, and the error bar indicates SD. White and black bars along the horizontal axes represent light and
dark periods, respectively. Numbers below the horizontal axes indicate the time in hours.
Regulation of FLOWERING LOCUS T by a MicroRNA
modulation of miR2032 was conserved among species in the
Pooideae. To address this question, we selected five Pooideae:
Dactylis glomerata, Bromus inermis Leyss, Agropyron cristatum,
Festuca arundinacea, and barley to compare miR2032 expression under SD and LD environments. As shown in Figure 7A, the
abundance of miR2032 in all five grasses was much higher when
they were grown under SDs than LDs, suggesting that photoperiodic regulation of miR2032 may have conserved roles in
flowering time control among Pooideae plants.
DISCUSSION
Plants have evolved sophisticated ways to restrict flowering to
the right time. As the most important flowering integrator, the
florigen FT has been the focus of attention for many plant scientists. Their findings about FT regulation so far have centered
on transcriptional regulation of FT; other layers of FT modulation,
11 of 15
such as mRNA and protein stability, remain a puzzle. Here, we
systemically characterized a newly evolved miR2032 in Pooideae
subfamily plants and found that miR2032 functions in photoperiodic regulation of flowering time via cleavage of transcripts of
FT-like genes.
MiRNAs function as key players in various aspects of plant
biological processes (Voinnet, 2009); however, research on plant
miRNA functions has mostly focused on conserved miRNAs,
and the biological behaviors of nonconserved miRNAs, especially in monocots, had remained largely unknown (Chuck et al.,
2009). The actions of miR2032 illustrated here may fill this vacancy to some extent. Even if FT genes have close relationships
in rice and B. distachyon, there is no miR2032 counterpart in rice
(Figure 1). This would not be a surprise, since Early heading
date1, a central gene essential for photoperiodic regulation of
flowering in rice, does not have an ortholog in B. distachyon
either (Doi et al., 2004; Higgins et al., 2010). These phenomena
Figure 7. Photoperiodic Regulation of MiR2032 Is Prevalent in Pooideae Plants.
(A) RNA gel blot analyses of miR2032 expressions in selected Pooideae grasses under SDs and LDs. Dg, D. glomerata; BiL, B. inermis Leyss; Ac,
A. cristatum; Fa, F. arundinacea; Hv, H. vulgare. U6 served as the RNA loading control of the RNA gel blot. Numbers below each miRNA gel blot
represent the fold changes relative to the miRNA under SDs.
(B) A working model for miR2032 in the photoperiodic flowering pathway in Pooideae plants. B. distachyon is used here as an example. When
B. distachyon grows under SDs, the repression of positive photoperiodic regulators (e.g., CO-like protein) as well as the activation of miR2032 lead to
low transcripts of florigen genes, FTL1/2, and the downstream gene VRN1 and thereby cause late flowering. When B. distachyon grows under LDs,
miR2032 is robustly inhibited, while the positive photoperiodic regulators (e.g., CO-like protein) are induced, resulting in high expression of FTL1/2,
VRN1, and early flowering. The question mark indicates that it remains to be further elucidated whether positive regulators (e.g., CO-like protein) in the
photoperiod pathway directly repress miR2032 activity under LDs. Lines with an arrow indicate positive regulation; lines with a vertical bar at the end
represent repressive regulation. Crosses (x) mean the breakdown of the pathway. V, vegetative stage; R, reproductive stage.
[See online article for color version of this figure.]
12 of 15
The Plant Cell
suggest that each plant species may have acquired its own
specific coding or noncoding RNAs to fine-tune the timing of
flowering.
Several lines of evidence assessed here indicate that there are
two florigen genes acting redundantly in B. distachyon: first,
FTL1 and FTL2 overexpressing transgenic plants flowered early
and arrested in the regeneration medium (see Supplemental
Figure 8 online); second, FTL1 and FTL2 show over 79% amino
acid sequence identity (see Supplemental Figure 4 and Supplemental Data Set 1 online); third, both FTL1 and FTL2 are
expressed outside the shoot apex and accumulate to much
higher levels in plants grown in LDs than in SDs (Figures 1 and
3); fourth, both FTL1 and FTL2 were predicted to miR2032 targets and their mRNA could be indeed cleaved by miR2032 in
vivo (Figure 1); fifth, the expression of FTL1 and FTL2 was severely reduced in miR2032 overexpression transgenic plants
(Figure 2); and sixth, both FTL1 and FTL2 transcripts accumulated to higher levels in MIM2032 than in the wild-type plants
under SDs (Figure 6). Nevertheless, differences between the two
genes may exist. For instance, compared with FTL2, the basepairing between miR2032 and FTL1 target site was not perfect,
and most FTL1 cleavage events did not occur in the canonical
miRNA cleavage site (Figure 1C), implying that miR2032 may
mediate FTL1 translation repression by a mechanism other than
mRNA degradation. Further isolation of single and double lossof-function mutants is necessary to dissect FTL1 and FTL2
functions in the B. distachyon flowering pathway.
The transition from vegetative to reproductive stage is determined by daylength in many plant varieties. In Arabidopsis,
CO is considered to be the central upstream regulator of FT
expression in the photoperiod flowering pathway (Turck et al.,
2008). Under LDs, both CO mRNA and protein are highly accumulated, thus resulting in activation of FT mRNA production
(Kobayashi and Weigel, 2007). In SDs, there is very little FT
expression because CO protein is largely degraded in a proteasome-dependent manner (Valverde et al., 2004). In Pooideae
plants, the newly evolved miR2032 could also directly regulate
FT transcripts. MiR2032 accumulates abundantly to repress FT
mRNAs in SD conditions, whereas miR2032 must, conversely,
be diminished for the induction of FT in LDs (Figures 3 and 4).
The different amounts of miR2032 with the daylength are partially due to the distinct chromatin conformations at pri-miRNA
genes, as we observed that the status of positive and negative
histone marks at MIR2032a and MIR2032b loci displayed profound differences under LDs and SDs (Figure 5).
Although the MIM2032 transgenic plants have a significant
effect on flowering time in SDs, they do not flower extremely
early like an LD plant. This is unlikely to be caused by the
flowering time variation in independent transgenic events
because the T1 segregated negative plants did not show any
early-heading performance compared with the wild type. Moreover, the MIM2032 plants did not display any early-flowering
phenotypes under LD conditions (see Supplemental Figure 9
online). These results convincingly suggest that miR2032 plays
a role in the photoperiod-mediated flowering regulation. However, we propose the possibility that the target mimic strategy
may not completely inhibit the activities of miR2032 in B. distachyon.
Further genetic studies to find knockout miR2032 plants are
essential for clearer understanding of miR2032 behaviors in
flowering control. Another possibility is that additional factors
other than miR2032 are also involved in photoperiod-mediated
floral induction. This is almost certain, since the dynamic
changes of FTL1 and FTL2 are not absolutely correlated with
miR2032 alteration in the shifting experiments (Figure 4). We
thereby propose a simple working model of the flowering
pathway in Pooideae plants involving miR2032 (Figure 7B). It
has been documented in wheat and barley that a pseudoresponse regulator protein named PPD1 functions upstream of CO
and is critical in the response to photoperiodic flowering control
(Turner et al., 2005; Shaw et al., 2012). Whether Pooideae
miR2032 acts downstream or functions in parallel with PPD1
and CO-like proteins in the photoperiod pathway remains to be
deciphered.
In all, our findings on miRNA-guided posttranscriptional
modulation of florigen gene contribute insights into the precise
regulatory modes of flowering in plants. Since most temperate
cereals, including barley and wheat, belong to the Pooideae
subfamily, artificial manipulation of miR2032 expression is perhaps a promising choice in crop breeding for proper heading
date.
METHODS
Plant Materials and Growth Conditions
For most experiments, the Brachypodium distachyon accession Bd21
was used. B. distachyon and other Pooideae grasses were grown under
SDs (8 h light/16 h dark) or LDs (16 h light/8 h dark) in growth chambers
with temperatures of 22°C during the day and 16°C at night. Three- to
four-week-old Bd21 seedlings harvested at Zeitgeber time 2 were used in
the experiments, unless stated otherwise.
Constructs and Plant Transformation
The 517-bp transcription unit MIR2032a sequence and FTLs coding
sequences were amplified from B. distachyon cDNA. FTLm was engineered by PCR-based mutation of miR2032 target site of FTL gene, and
the miR2032 target mimic sequence was amplified from Arabidopsis
thaliana and artificially modified as described before (Todesco et al.,
2010). All these genes and target mimic constructs were under the control
of maize (Zea mays) UBIQUITIN promoter for constitutive expression in B.
distachyon plants. The sequences of primers for B. distachyon FT gene
cDNA, miR2032 precursor amplification, and target mimic constructs are
listed in Supplemental Table 1 online. Agrobacterium tumefaciens–mediated
transformation of B. distachyon was essentially as previously described
(Alves et al., 2009).
RNA Expression Analysis
Total RNA was extracted with Trizol reagent (Invitrogen) from three pooled
B. distachyon plants grown under the indicated conditions. Thirty micrograms of enriched sRNAs were used for RNA gel blot detection of
miR2032 amounts. 32P-end-labeled oligonucleotides complementary to
miRNA sequences were used as probes. Total RNA was pretreated with
RNase-free DNase I (Promega) to remove DNA and then reverse transcribed by EasyScript reverse transcriptase (TransGen Biotech) with oligo
(dT) primers. RT-PCR and qPCR analyses of gene transcripts were
performed as previously reported (Wu et al., 2010). qPCR was performed
with SYBR Premix EX Taq (Takara). GAPDH mRNA was detected in
Regulation of FLOWERING LOCUS T by a MicroRNA
parallel and used for data normalization. RT-PCR analyses for pri-miR2032,
target gene expressions, and RNA gel blots for mature miR2032 were
performed at least two times with similar results. qRT-PCRs were performed
from three biological replicates with three technique repeats. Sequences of
all probes and primers are listed in Supplemental Table 1 online.
RACE Mapping of MIRNA Primary Transcripts and Target
Cleavage Site
59 RNA ligase-mediated RACE reactions for pri-miR2032 transcript 59-end
determination were performed using poly(A)-enriched RNA that was
pretreated with calf intestine phosphatase plus tobacco acid pyrophosphatase, and the cDNA was synthesized using random oligonucleotides as primers (Ambion). For 39 MIRNA primary transcripts, cDNA
was synthesized using adaptor-tagged oligo(dT) primers. 59 RACE of
miR2032 target sites of FT genes was performed using poly(A) -enriched
RNA without calf intestine phosphatase plus tobacco acid pyrophosphatase treatment. All amplification products were gel purified and cloned
in the pGEM-T easy vector (Promega) for sequencing.
DNA Methylation and Histone Modification Analyses
Genomic DNA was isolated from 3-week-old B. distachyon seedlings grown
in SDs and LDs using the DNA secure plant kit (Tiangen). Bisulfite sequencing and PCR-based DNA methylation assays were conducted as
described previously (Wu et al., 2010). The PCR products were cloned into
the pGEM-T easy vector (Promega), and at least 20 individual clones were
sequenced. ChIP was performed as described before with minor modifications. Two grams of 3-week-old B. distachyon tissue was treated with
formaldehyde buffer for 20 min at room temperature for cross-linking by
vacuum infiltration, followed by addition of 125 mM Gly. Plants were rinsed
with water, frozen, and ground into powder in liquid nitrogen using a mortar
and pestle, suspended in 30 mL of Extraction Buffer I (10 mM Tris-HCl, pH
8.0, 0.5 M Suc, 10 mM MgCl2, 5 mM DTT, 1 mM PMSF, and 1% Roche
protease inhibitors), and filtered through two layers of Miracloth and
centrifuged at 3000g at 4°C for 15 min to remove debris. Nuclear pellets
were resuspended in 1 mL of Extraction Buffer II (10 mM Tris-HCl, pH 8.0,
0.5 M Suc, 10 mM MgCl2, 0.5% Triton X-100, 5 mM DTT, 1 mM PMSF, and
1% tablet Roche protease inhibitors) and centrifuged at 12,000g at 4°C for
10 min. Then the pellets were suspended in nuclei lysis buffer (50 mM TrisHCl, pH 8.0, 10 mM EDTA, 1% SDS, 5 mM DTT, and 1% Roche protease
inhibitors) and sonicated five times for 20 s each. After centrifugation at
16,000g for 10 min, the supernatant was diluted 10-fold with ChIP dilution
buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, and
167 mM NaCl). After being precleared for 1 h, 25 mL of protein A agarose
(Roche) and the appropriate antibodies were added for incubation overnight. Agarose-antibody complexes were washed five times, 5 min each,
with low-salt, high-salt, LiCl washing buffer, and TE buffer. Elution and
reverse cross-linking were performed as described previously (Saleh et al.,
2008). One microliter of extracted DNA was used as template to amplify the
indicated sequences for histone modification status.
Sequence Alignment and Phylogenetic Analysis
BLASTP was performed using the deduced amino acid sequences of FT
genes from B. distachyon, Arabidopsis thaliana, and barley (Hordeum
vulgare). Multiple sequence alignment was performed using ClustalW,
and the construction of phylogenetic tree was conducted by MEGA
program using the neighbor-joining method. Bootstrap analysis was
performed to estimate nodal support on the basis of 1000 replicates.
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under the following accession numbers: FTL1, Bradi2g07070; FTL2,
Bradi1g48830; FTL4, Bradi4g35040; FTL5, Bradi5g14010; FTL6,
Bradi3g48036; FTL7a, Bradi4g39730; FTL7b, Bradi4g39750; FTL7c,
Bradi4g39760; FTL9, Bradi2g49795; FTL10, Bradi2g19670; FTL12,
Bradi1g38150; FTL13, Bradi3g08890; VRN1, Bradi3g08340; FUL2,
Bradi1g59250; DCL1, Bradi1g77090; MIR2032a (1-14), KF638635-638648;
MIR2032b, KF638649; Arabidopsis FT, At1g15480; Arabidopsis TSF,
At4g20370; barley FT1, DQ100327; barley FT2, DQ297407; barley FT3,
DQ411319; barley FT4, DQ411320; and barley FT5, EF012202.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. The Flowering Habit of Brachypodium
Accession Bd21.
Supplemental Figure 2. Conservation of miR2032 in Pooideae Plants.
Supplemental Figure 3. Gene Structures of Pri-miR2032a and
Pri-miR2032b.
Supplemental Figure 4. FT-Like Orthologous Genes Are Potential
MiR2032 Targets in Wheat and Barley.
Supplemental Figure 5. Effects of MiR2032 Ectopic Expression on
Flowering Time and Plant Height.
Supplemental Figure 6. Transcriptional Control of MiR2032 by
Photoperiod.
Supplemental Figure 7. DNA Methylation Analysis at Pri-miR2032
Loci in Brachypodium Grown under SDs and LDs.
Supplemental Figure 8. Extremely Early Flowering Phenotypes of
Transgenic Plants Ectopically Expressing Either FTL1 or FTL2 Gene
with or without MiR2032 Target Sites.
Supplemental Figure 9. MiR2032 Target Mimicry Specifically Alters
Flowering Time in Transgenic Plants under SD Conditions.
Supplemental Table 1. Oligos Used as Primers and Probes in This
Study.
Supplemental Data Set 1. Text File of Alignment Corresponding to
Phylogenetic Analysis in Supplemental Figure 4.
ACKNOWLEDGMENTS
We thank Lihui Li, Yuqing Lu, and Lihong Miao for providing the seeds of
Pooideae grasses. We also thank Yijun Qi, Jiawei Wang, and Detlef
Weigel for critical reading of the article. This work was supported in part
by the National Natural Science Foundation of China (31200181 and
30871323), the National Basic Research “973” (2009CB118306 and
2010CB125902), and a Core Research Budget of the Non-profit
Governmental Research Institution (Institute of Crop Science, Chinese
Academy of Agricultural Sciences).
AUTHOR CONTRIBUTIONS
L.W. and L.M. designed the research. L.W., Do.L., J.W., Z.Q., R.Z., and
Da.L. performed the experiments. L.W., Do.L., A.L., D.F., W.Z., and L.M.
analyzed the data. L.W. and L.M. wrote the article. L.W., W.Z., and L.M.
contributed equally to this work.
Accession Numbers
Sequence data from this article can be found in the Brachypodium
Genome Initiative, Arabidopsis Genome Initiative, or GenBank databases
Received September 17, 2013; revised October 24, 2013; accepted
November 6, 2013; published November 27, 2013.
14 of 15
The Plant Cell
REFERENCES
Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A.,
Ikeda, Y., Ichinoki, H., Notaguchi, M., Goto, K., and Araki, T.
(2005). FD, a bZIP protein mediating signals from the floral pathway
integrator FT at the shoot apex. Science 309: 1052–1056.
Abrouk, M., Zhang, R., Murat, F., Li, A., Pont, C., Mao, L., and
Salse, J. (2012). Grass microRNA gene paleohistory unveils new
insights into gene dosage balance in subgenome partitioning after
whole-genome duplication. Plant Cell 24: 1776–1792.
Adrian, J., Farrona, S., Reimer, J.J., Albani, M.C., Coupland, G., and
Turck, F. (2010). cis-regulatory elements and chromatin state
coordinately control temporal and spatial expression of FLOWERING
LOCUS T in Arabidopsis. Plant Cell 22: 1425–1440.
Alves, S.C., Worland, B., Thole, V., Snape, J.W., Bevan, M.W., and
Vain, P. (2009). A protocol for Agrobacterium-mediated transformation
of Brachypodium distachyon community standard line Bd21. Nat.
Protoc. 4: 638–649.
Bastow, R., Mylne, J.S., Lister, C., Lippman, Z., Martienssen, R.A.,
and Dean, C. (2004). Vernalization requires epigenetic silencing of
FLC by histone methylation. Nature 427: 164–167.
Bäurle, I., and Dean, C. (2006). The timing of developmental transitions in
plants. Cell 125: 655–664.
Bergonzi, S., Albani, M.C., Ver Loren van Themaat, E., Nordström, K.J.,
Wang, R., Schneeberger, K., Moerland, P.D., and Coupland, G.
(2013). Mechanisms of age-dependent response to winter temperature
in perennial flowering of Arabis alpina. Science 340: 1094–1097.
Brkljacic, J., et al. (2011). Brachypodium as a model for the grasses:
Today and the future. Plant Physiol. 157: 3–13.
Castillejo, C., and Pelaz, S. (2008). The balance between CONSTANS and
TEMPRANILLO activities determines FT expression to trigger flowering.
Curr. Biol. 18: 1338–1343.
Chen, H.M., Chen, L.T., Patel, K., Li, Y.H., Baulcombe, D.C., and Wu, S.H.
(2010). 22-Nucleotide RNAs trigger secondary siRNA biogenesis in plants.
Proc. Natl. Acad. Sci. USA 107: 15269–15274.
Chuck, G., Candela, H., and Hake, S. (2009). Big impacts by small
RNAs in plant development. Curr. Opin. Plant Biol. 12: 81–86.
Corbesier, L., Vincent, C., Jang, S., Fornara, F., Fan, Q., Searle, I.,
Giakountis, A., Farrona, S., Gissot, L., Turnbull, C., and Coupland, G.
(2007). FT protein movement contributes to long-distance signaling in
floral induction of Arabidopsis. Science 316: 1030–1033.
Cuperus, J.T., Carbonell, A., Fahlgren, N., Garcia-Ruiz, H., Burke,
R.T., Takeda, A., Sullivan, C.M., Gilbert, S.D., Montgomery, T.A.,
and Carrington, J.C. (2010). Unique functionality of 22-nt miRNAs
in triggering RDR6-dependent siRNA biogenesis from target
transcripts in Arabidopsis. Nat. Struct. Mol. Biol. 17: 997–1003.
Distelfeld, A., Li, C., and Dubcovsky, J. (2009). Regulation of flowering in
temperate cereals. Curr. Opin. Plant Biol. 12: 178–184.
Doi, K., Izawa, T., Fuse, T., Yamanouchi, U., Kubo, T., Shimatani, Z.,
Yano, M., and Yoshimura, A. (2004). Ehd1, a B-type response regulator
in rice, confers short-day promotion of flowering and controls FT-like
gene expression independently of Hd1. Genes Dev. 18: 926–936.
Farrona, S., Coupland, G., and Turck, F. (2008). The impact of
chromatin regulation on the floral transition. Semin. Cell Dev. Biol.
19: 560–573.
Filiz, E., Ozdemir, B.S., Budak, F., Vogel, J.P., Tuna, M., and Budak,
H. (2009). Molecular, morphological, and cytological analysis of diverse
Brachypodium distachyon inbred lines. Genome 52: 876–890.
Fornara, F., de Montaigu, A., and Coupland, G. (2010). SnapShot:
Control of flowering in Arabidopsis. Cell 141: 550, e1–e2.
Franco-Zorrilla, J.M., Valli, A., Todesco, M., Mateos, I., Puga, M.I.,
Rubio-Somoza, I., Leyva, A., Weigel, D., García, J.A., and PazAres, J. (2007). Target mimicry provides a new mechanism for
regulation of microRNA activity. Nat. Genet. 39: 1033–1037.
Hackenberg, M., Shi, B.J., Gustafson, P., and Langridge, P. (2012).
A transgenic transcription factor (TaDREB3) in barley affects the
expression of microRNAs and other small non-coding RNAs. PLoS
ONE 7: e42030.
Higgins, J.A., Bailey, P.C., and Laurie, D.A. (2010). Comparative
genomics of flowering time pathways using Brachypodium distachyon
as a model for the temperate grasses. PLoS ONE 5: e10065.
Hong, S.Y., Lee, S., Seo, P.J., Yang, M.S., and Park, C.M. (2010).
Identification and molecular characterization of a Brachypodium
distachyon GIGANTEA gene: Functional conservation in monocot
and dicot plants. Plant Mol. Biol. 72: 485–497.
Kardailsky, I., Shukla, V.K., Ahn, J.H., Dagenais, N., Christensen,
S.K., Nguyen, J.T., Chory, J., Harrison, M.J., and Weigel, D. (1999).
Activation tagging of the floral inducer FT. Science 286: 1962–1965.
Kim, J.J., Lee, J.H., Kim, W., Jung, H.S., Huijser, P., and Ahn, J.H.
(2012). The microRNA156-SQUAMOSA PROMOTER BINDING
PROTEIN-LIKE3 module regulates ambient temperature-responsive
flowering via FLOWERING LOCUS T in Arabidopsis. Plant Physiol.
159: 461–478.
Kobayashi, Y., Kaya, H., Goto, K., Iwabuchi, M., and Araki, T.
(1999). A pair of related genes with antagonistic roles in mediating
flowering signals. Science 286: 1960–1962.
Kobayashi, Y., and Weigel, D. (2007). Move on up, it’s time for change—
Mobile signals controlling photoperiod-dependent flowering. Genes
Dev. 21: 2371–2384.
Komiya, R., Yokoi, S., and Shimamoto, K. (2009). A gene network for
long-day flowering activates RFT1 encoding a mobile flowering
signal in rice. Development 136: 3443–3450.
Li, C., and Dubcovsky, J. (2008). Wheat FT protein regulates VRN1
transcription through interactions with FDL2. Plant J. 55: 543–554.
Li, D., Liu, C., Shen, L., Wu, Y., Chen, H., Robertson, M., Helliwell, C.A.,
Ito, T., Meyerowitz, E., and Yu, H. (2008). A repressor complex governs
the integration of flowering signals in Arabidopsis. Dev. Cell 15:
110–120.
Lifschitz, E., Eviatar, T., Rozman, A., Shalit, A., Goldshmidt, A.,
Amsellem, Z., Alvarez, J.P., and Eshed, Y. (2006). The tomato FT
ortholog triggers systemic signals that regulate growth and flowering
and substitute for diverse environmental stimuli. Proc. Natl. Acad. Sci.
USA 103: 6398–6403.
Lucas, S.J., and Budak, H. (2012). Sorting the wheat from the chaff:
Identifying miRNAs in genomic survey sequences of Triticum
aestivum chromosome 1AL. PLoS ONE 7: e40859.
Manavella, P.A., Koenig, D., and Weigel, D. (2012). Plant secondary
siRNA production determined by microRNA-duplex structure. Proc.
Natl. Acad. Sci. USA 109: 2461–2466.
Mathieu, J., Warthmann, N., Küttner, F., and Schmid, M. (2007).
Export of FT protein from phloem companion cells is sufficient for
floral induction in Arabidopsis. Curr. Biol. 17: 1055–1060.
Mathieu, J., Yant, L.J., Mürdter, F., Küttner, F., and Schmid, M.
(2009). Repression of flowering by the miR172 target SMZ. PLoS
Biol. 7: e1000148.
Meng, X., Muszynski, M.G., and Danilevskaya, O.N. (2011). The FTlike ZCN8 gene functions as a floral activator and is involved in
photoperiod sensitivity in maize. Plant Cell 23: 942–960.
Navarro, C., Abelenda, J.A., Cruz-Oró, E., Cuéllar, C.A., Tamaki, S.,
Silva, J., Shimamoto, K., and Prat, S. (2011). Control of flowering
and storage organ formation in potato by FLOWERING LOCUS T.
Nature 478: 119–122.
Opanowicz, M., Vain, P., Draper, J., Parker, D., and Doonan, J.H.
(2008). Brachypodium distachyon: Making hay with a wild grass.
Trends Plant Sci. 13: 172–177.
Pin, P.A., Benlloch, R., Bonnet, D., Wremerth-Weich, E., Kraft, T.,
Gielen, J.J., and Nilsson, O. (2010). An antagonistic pair of FT
Regulation of FLOWERING LOCUS T by a MicroRNA
homologs mediates the control of flowering time in sugar beet.
Science 330: 1397–1400.
Saleh, A., Alvarez-Venegas, R., and Avramova, Z. (2008). An efficient
chromatin immunoprecipitation (ChIP) protocol for studying histone
modifications in Arabidopsis plants. Nat. Protoc. 3: 1018–1025.
Sawa, M., and Kay, S.A. (2011). GIGANTEA directly activates Flowering
Locus T in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 108:
11698–11703.
Schreiber, A.W., Shi, B.J., Huang, C.Y., Langridge, P., and
Baumann, U. (2011). Discovery of barley miRNAs through deep
sequencing of short reads. BMC Genomics 12: 129.
Schwartz, C.J., Doyle, M.R., Manzaneda, A.J., Rey, P.J., MitchellOlds, T., and Amasino, R.M. (2010). Natural variation of flowering
time and vernalization responsiveness in Brachypodium distachyon.
Bioenerg. Res. 3: 38–46.
Seo, E., Yu, J., Ryu, K.H., Lee, M.M., and Lee, I. (2011). WEREWOLF,
a regulator of root hair pattern formation, controls flowering time through
the regulation of FT mRNA stability. Plant Physiol. 156: 1867–1877.
Shaw, L.M., Turner, A.S., and Laurie, D.A. (2012). The impact of
photoperiod insensitive Ppd-1a mutations on the photoperiod
pathway across the three genomes of hexaploid wheat (Triticum
aestivum). Plant J. 71: 71–84.
Suárez-López, P., Wheatley, K., Robson, F., Onouchi, H., Valverde,
F., and Coupland, G. (2001). CONSTANS mediates between the
circadian clock and the control of flowering in Arabidopsis. Nature
410: 1116–1120.
Tamaki, S., Matsuo, S., Wong, H.L., Yokoi, S., and Shimamoto, K. (2007).
Hd3a protein is a mobile flowering signal in rice. Science 316: 1033–1036.
Todesco, M., Rubio-Somoza, I., Paz-Ares, J., and Weigel, D. (2010). A
collection of target mimics for comprehensive analysis of microRNA
function in Arabidopsis thaliana. PLoS Genet. 6: e1001031.
Torti, S., Fornara, F., Vincent, C., Andrés, F., Nordström, K., Göbel,
U., Knoll, D., Schoof, H., and Coupland, G. (2012). Analysis of the
Arabidopsis shoot meristem transcriptome during floral transition
identifies distinct regulatory patterns and a leucine-rich repeat
protein that promotes flowering. Plant Cell 24: 444–462.
Trevaskis, B., Bagnall, D.J., Ellis, M.H., Peacock, W.J., and Dennis,
E.S. (2003). MADS box genes control vernalization-induced
flowering in cereals. Proc. Natl. Acad. Sci. USA 100: 13099–13104.
Turck, F., Fornara, F., and Coupland, G. (2008). Regulation and
identity of florigen: FLOWERING LOCUS T moves center stage.
Annu. Rev. Plant Biol. 59: 573–594.
Turner, A., Beales, J., Faure, S., Dunford, R.P., and Laurie, D.A.
(2005). The pseudo-response regulator Ppd-H1 provides adaptation to
photoperiod in barley. Science 310: 1031–1034.
Valverde, F., Mouradov, A., Soppe, W., Ravenscroft, D., Samach, A.,
and Coupland, G. (2004). Photoreceptor regulation of CONSTANS
protein in photoperiodic flowering. Science 303: 1003–1006.
15 of 15
Vogel, J.P., et al; International Brachypodium Initiative (2010).
Genome sequencing and analysis of the model grass Brachypodium
distachyon. Nature 463: 763–768.
Voinnet, O. (2009). Origin, biogenesis, and activity of plant microRNAs.
Cell 136: 669–687.
Wang, J.W., Czech, B., and Weigel, D. (2009). MiR156-regulated SPL
transcription factors define an endogenous flowering pathway in
Arabidopsis thaliana. Cell 138: 738–749.
Wei, B., Cai, T., Zhang, R., Li, A., Huo, N., Li, S., Gu, Y.Q., Vogel, J.,
Jia, J., Qi, Y., and Mao, L. (2009). Novel microRNAs uncovered by
deep sequencing of small RNA transcriptomes in bread wheat
(Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv.
Funct. Integr. Genomics 9: 499–511.
Wenkel, S., Turck, F., Singer, K., Gissot, L., Le Gourrierec, J.,
Samach, A., and Coupland, G. (2006). CONSTANS and the CCAAT
box binding complex share a functionally important domain and interact
to regulate flowering of Arabidopsis. Plant Cell 18: 2971–2984.
Wigge, P.A. (2011). FT, a mobile developmental signal in plants. Curr.
Biol. 21: R374–R378.
Wigge, P.A., Kim, M.C., Jaeger, K.E., Busch, W., Schmid, M.,
Lohmann, J.U., and Weigel, D. (2005). Integration of spatial and
temporal information during floral induction in Arabidopsis. Science
309: 1056–1059.
Wu, G., Park, M.Y., Conway, S.R., Wang, J.W., Weigel, D., and
Poethig, R.S. (2009a). The sequential action of miR156 and miR172
regulates developmental timing in Arabidopsis. Cell 138: 750–759.
Wu, H.J., Ma, Y.K., Chen, T., Wang, M., and Wang, X.J. (2012).
PsRobot: A web-based plant small RNA meta-analysis toolbox.
Nucleic Acids Res. 40 (Web Server issue): W22–W28.
Wu, L., Zhang, Q., Zhou, H., Ni, F., Wu, X., and Qi, Y. (2009b). Rice
microRNA effector complexes and targets. Plant Cell 21: 3421–3435.
Wu, L., Zhou, H., Zhang, Q., Zhang, J., Ni, F., Liu, C., and Qi, Y.
(2010). DNA methylation mediated by a microRNA pathway. Mol.
Cell 38: 465–475.
Xie, Z., Allen, E., Fahlgren, N., Calamar, A., Givan, S.A., and
Carrington, J.C. (2005). Expression of Arabidopsis MIRNA genes.
Plant Physiol. 138: 2145–2154.
Yan, L., Fu, D., Li, C., Blechl, A., Tranquilli, G., Bonafede, M.,
Sanchez, A., Valarik, M., Yasuda, S., and Dubcovsky, J. (2006).
The wheat and barley vernalization gene VRN3 is an orthologue of
FT. Proc. Natl. Acad. Sci. USA 103: 19581–19586.
Zhang, J., Xu, Y., Huan, Q., and Chong, K. (2009). Deep sequencing
of Brachypodium small RNAs at the global genome level identifies
microRNAs involved in cold stress response. BMC Genomics 10: 449.
Zhou, C.M., Zhang, T.Q., Wang, X., Yu, S., Lian, H., Tang, H., Feng,
Z.Y., Zozomova-Lihová, J., and Wang, J.W. (2013). Molecular
basis of age-dependent vernalization in Cardamine flexuosa.
Science 340: 1097–1100.
Regulation of FLOWERING LOCUS T by a MicroRNA in Brachypodium distachyon
Liang Wu, Dongfeng Liu, Jiajie Wu, Rongzhi Zhang, Zhengrui Qin, Danmei Liu, Aili Li, Daolin Fu,
Wenxue Zhai and Long Mao
Plant Cell; originally published online November 27, 2013;
DOI 10.1105/tpc.113.118620
This information is current as of June 15, 2017
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