Laboratoire de Biologie Cellulaire, Institut J. P. Bourgin, INRA, 78026 Versailles Cedex, France
* Author for correspondence (e-mail: laufs{at}versailles.inra.fr)
Accepted 22 June 2004
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SUMMARY |
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Key words: MicroRNA, Meristem, Boundary, Cell fate, Differentiation, Proliferation
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Introduction |
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MiRNAs are small, single-stranded RNAs of about 21 nucleotides found in
both animals and plants that post-transcriptionally regulate gene expression
(for reviews, see Bartel, 2004;
Lai, 2003
). Animal miRNAs are
transcribed as long primary transcripts (pri-miRNAs) that are first processed
into hairpin precursors of about 70 nucleotides (pre-miRNAs) and then into
mature miRNAs. Although the cleavage affects both strands of the hairpin
precursors, only one strand, the mature miRNA, is preferentially accumulated
and incorporated into a ribonucleoprotein complex, the miRNP complex
(Khvorova et al., 2003
;
Mourelatos et al., 2002
;
Schwarz et al., 2003
).
Interaction of the miRNA with imperfect complementary sequences located in the
3' untranslated region (UTR) of the target mRNAs leads to translational
attenuation. Conversely, plant miRNAs are perfectly or almost perfectly
complementary to their targets (Rhoades et
al., 2002
), and their interaction triggers the cleavage of the
mRNA (Han et al., 2004
;
Kasschau et al., 2003
;
Llave et al., 2002
;
Palatnik et al., 2003
;
Tang et al., 2003
;
Vazquez et al., 2004
;
Xie et al., 2003
), although
examples of translational attenuation have also been reported
(Aukerman and Sakai, 2003
;
Chen, 2004
).
In animals, most evidence for miRNA regulation of gene expression results
from classical genetic approaches, although potential targets of miRNAs have
been recently predicted by bioinformatics
(Enright et al., 2003;
Lewis et al., 2003
;
Rajewsky and Socci, 2004
).
miRNAs were first identified as regulators of the developmental timing in
C. elegans (Abrahante et al.,
2003
; Lin et al.,
2003
; Reinhart et al.,
2000
; Slack et al.,
2000
). Additional evidence suggests that they may be involved in
spatial patterning processes. For example, left-right asymmetry during
neuronal patterning in C. elegans is controlled by a miRNA
(Johnston and Hobert, 2003
).
In Drosophila, the Bantam miRNA, the expression of which
responds to patterning cues, promotes cell proliferation and prevents
apoptosis by targeting the pro-apoptotic gene hid
(Brennecke et al., 2003
). Thus,
Bantam may participate in the coordination between patterning events
and downstream control of cell death and cell proliferation.
In plants, most of the miRNA targets predicted by bioinformatics are
transcription factors involved in the control of development, raising the
possibility that miRNAs may play an important role in this process
(Rhoades et al., 2002).
Organogenesis in plants, in contrast to animals, proceeds throughout their
life span as new tissues and organs are continuously produced by meristems.
For example, the shoot apical meristem and a related structure, the floral
meristem, initiate primordia of lateral organs such as leaves, sepals or
stamens. A family of miRNA, miR172 negatively regulates
APETALA2-like transcription factors, thus controlling flowering time
and floral organ identity (Aukerman and
Sakai, 2003
; Chen,
2004
). Another miRNA family, JAW/miR159, which negatively
regulates several members of the TCP and MYB transcription factor families is
involved in leaf development (Palatnik et
al., 2003
). miRNAs have also a central role in lateral organ
polarisation. Lateral organ polarity is controlled by three members of a
homeodomain/leucine zipper transcription factor family, PHABULOSA
(PHB), PHAVULOTA (PHV) and REVOLUTA
(REV) (Emery et al.,
2003
; McConnell et al.,
2001
; Otsuga et al.,
2001
). These genes and two evolutionary conserved miRNA,
miR165 and miR166 predicted to target them, are expressed in
complementary domains of the developing lateral organs
(Juarez et al., 2004
;
Kidner and Martienssen, 2004
).
miRNA-resistant forms of these targets are ectopically expressed in the
developing primordia, suggesting that miRNAs normally limit their expression
pattern (Emery et al., 2003
;
Juarez et al., 2004
;
Kidner and Martienssen, 2004
;
McConnell et al., 2001
;
Tang et al., 2003
).
Here we have analysed the role of a miRNA, miR164, in the
regulation of the boundary domain around developing primordia at the shoot
apical and floral meristems. Boundary establishment and maintenance is
controlled in Arabidopsis by three partially redundant genes,
CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and
CUC3 (Aida et al.,
1997; Aida et al.,
1999
; Takada et al.,
2001
; Vroemen et al.,
2003
). These three members of the NAC transcription factor family
are expressed in the cells forming the boundary domain around primordia where
they may repress growth (Aida et al.,
1999
). A single mutation of either CUC gene has no major effect on
boundary formation, whereas double mutants have fused cotyledons reflecting
abnormal boundary specification during embryo development
(Aida et al., 1997
;
Vroemen et al., 2003
). Later
on, the cuc1 cuc2 double mutant phenotype is restricted to the
flowers that form partially fused organs
(Aida et al., 1997
). The
absence of a mutant phenotype during the vegetative phase and in the
inflorescence stem was proposed to be due to partial redundancy between the
three CUC genes identified in Arabidopsis. In addition to their role
in boundary specification, the CUC genes are also involved in meristem
establishment during embryogenesis. Indeed, the CUC genes promote the
expression of the SHOOT MERISTEMLESS (STM) gene, a central
determinant of meristem identity (Daimon
et al., 2003
; Hibara et al.,
2003
; Takada et al.,
2001
; Vroemen et al.,
2003
). Sequence homology suggests that CUC1 and
CUC2 mRNAs could be targeted by miR164
(Rhoades et al., 2002
).
Accordingly, their expression levels are increased in Arabidopsis
backgrounds with an impaired miRNA pathway
(Kasschau et al., 2003
;
Vazquez et al., 2004
). We show
that miR164 targets CUC1 and CUC2 but not
CUC3 mRNAs for degradation, in planta. Disruption of CUC2
regulation by the miR164, either by making it resistant to the miRNA
or by reducing the miRNA level leads to a similar boundary enlargement
phenotype. We traced this modification back to the proliferative activity of
the boundary cells. Therefore, we propose a model where miR164
mediates the degradation of CUC1 and CUC2 mRNAs, and thus
limits the expansion of the boundary domain.
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Materials and methods |
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Plant material
Plants were transformed by floral-dip
(Clough and Bent, 1998).
AlcA::CUC2 STM::ALCR alcA::erGFP lines were generated by
retransforming a STM::ALCR alcA::erGFP line. The LFY::ALCR
alcA::GUS and LFY::ALCR alcA::GUS alcAH4GFP have been described
previously (Deveaux et al.,
2003
). The M0223 enhancer trap line described by Cary et al.
(Cary et al., 2002
) comes from
the Hasselhoff collection and was provided by the Nottingham Arabidopsis
Biological Stock Centre. The hyl1-1
(Lu and Fedoroff, 2000
) and
hen1-5 (Vazquez et al.,
2004
) mutants were kindly provided by N. Fedoroff and H.
Vaucheret, respectively, and the dcl1-9 mutant was provided by the
NASC.
Plant growth in vitro or in the greenhouse, and ethanol induction in the
greenhouse have been described before
(Deveaux et al., 2003) with
the exception that 75% ethanol was used for vapour induction instead of 95%.
For in vitro induction, 0.1% of ethanol was added to the growth media before
pouring the plates.
RNA analysis
Total RNA was extracted from inflorescence apices using TRIZOL (Invitrogen)
according to manufacturer's instructions.
For miRNA detection, 30 µg of total RNA were separated overnight on a 15% acrylamide, 8 M urea gel and blotted on Hybond-NX membranes using a BioRad semi-dry blotter. Filters were hybridised overnight in Church buffer at 30°C with end-labelled primers, then washed for 1 hour in 2xSSC, 0.1% SDS. Blots were reprobed with a 5S RNA probe.
For HMW RNA, 20 µg of total RNA were separated on a 1.5% agarose gel, blotted on nylon membranes and probed with a randomly 32P-labelled DNA fragment specific for CUC2 (from 415 bp after the ATG to the STOP codon).
RT-PCR was carried out as previously described
(Laufs et al., 2003) using
primers located on two different exons to discriminate between genomic
contamination and RT products. Furthermore, the two primers, located on each
side of the predicted miR164 cleavage site, selectively amplified
only the uncleaved mRNA. Twenty-three PCR cycles were run for CUC1;
21 cycles for NAC1 and At5g07680; 19 cycles for
At5g61430, CUC2 and CUC3; and 15 cycles for APT. The primers
used are indicated in Table
1.
|
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Results |
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In summary, 2x35S::miR164A and 2x35S::miR164B lines exhibited embryo patterning and floral defects that are characteristic for Arabidopsis lines with reduced CUC1 and/or CUC2 activity.
Northern blot analysis using a probe complementary to miR164
revealed a small RNA of 21-22 nucleotide whose level was increased in the
2x35S::miR164 lines compared with wild-type or 2x35S::erGFP
plants and correlated with the phenotype intensity of the
2x35S::miR164 lines (Fig.
1D, parts 1,2). We next checked if the RNA species we detected
corresponded to a single-stranded miRNA or to a double-stranded siRNA. To
achieve this, we performed additional northern blots to detect
miR164A* and miR164B*, the
complementary strands to miR164 that result from RNaseIII processing
of miR164A and miR164B precursors, respectively
(Fig. 1D, parts 1,3). The
accumulation level of miR164B* is below detection level in
both wild-type and 2x35S::miR164B plants
(Fig. 1D, part 3). Whereas
miR164A* could not be detected in wild-type plants, it
accumulated in the 2x35S::miR164A line, though at a
10-20 times
lower level than miR164 (Fig.
1D, part 3). A similar low-level of the
miR164A* strand has been reported previously for plant
(Reinhart et al., 2002
) or
animal miRNAs (Lagos-Quintana et al.,
2003
; Lagos-Quintana et al.,
2002
; Lau et al.,
2001
; Lim et al.,
2003
; Mourelatos et al.,
2002
). We therefore concluded that bona fide miR164
accumulated in the 2x35S::miR164 lines.
In conclusion, miR164 overexpression phenocopied the cuc1 cuc2 double mutants and the severity of the phenotype correlated with the level of miR164 accumulation.
miR164 primarily targets four genes of the NAC family
miR164 was predicted by Rhoades et al.
(Rhoades et al., 2002) to
target 5 members of the NAC gene family: CUC1 and CUC2, NAC1
(At1g56010) that has been implicated in lateral root development
(Xie et al., 2000
) and two
other uncharacterised members (At5g07680 and At5g61430). We
analysed by RT-PCR the effects of miR164 overexpression on the steady
state accumulation levels of the five predicted targets and of CUC3,
a gene partially redundant with CUC1 and CUC2 but lacking a
miR164-binding site.
CUC1 and CUC2 mRNA levels in 2x35S::miR164 lines
were reduced compared with wild-type plants
(Fig. 2A). The reduction could
reach 90% of the wild-type level and correlated with the intensity of the
floral defect phenotype. Although the CUC3 mRNA accumulation was
reduced in the strong lines, the amplitude was lower compared with
CUC1 and CUC2. It has been reported that the expression of
CUC3 is abolished or reduced in the absence of both CUC1 and CUC2
activities (Vroemen et al.,
2003), showing that CUC1 and CUC2 are redundantly required for
CUC3 expression. Therefore, downregulation of CUC3 in
miR164 overexpressers is likely to be a secondary effect of
CUC1 and CUC2 inactivation. The absence of a region
complementary to miR164 in CUC3 also supports the hypothesis
that CUC3 is not a direct target of miR164.
|
miR164 regulation of CUC2 is essential for plant development
In order to assess in planta the importance of miR164-guided
cleavage of CUC2, we modified the CUC2 mRNA to make it
potentially resistant to miR164-guided cleavage, without altering the
protein sequence. To achieve this, we first introduced four mismatches in the
miR164-binding site of CUC2 in addition to the three
naturally present (CUC2-m4 in Fig.
3A) and ubiquitously overexpressed this modified CUC2 or
the wild-type form using the double 35S promoter in transgenic WS
Arabidopsis (Fig. 3B).
Most of the lines had wrinkled leaves, regardless of the CUC2 form
overexpressed (9/11 2x35S::CUC2 lines and 7/7 2x35S::CUC2-m4
lines). Inflorescence size was reduced in six 2x35S::CUC2 lines. A
similar phenotype was observed in three 2x35S::CUC2-m4
lines. In addition, two CUC2-m4 lines showed a more severe phenotype
with extreme reduction of internode elongation, small floral organs and
reduced fertility. Reduced growth was reported for transgenic lines expressing
CUC1 that, in addition, showed ectopic meristems on the cotyledon
surface (Hibara et al., 2003;
Takada et al., 2001
). We did
not observe ectopic meristems, reflecting either different effects of
CUC1 and CUC2 or specific response of the ecotypes used as
CUC1 overexpressers were in Ler background. The more severe phenotype
of the 2x35S::CUC2-m4 lines suggested that miR164 regulation
was important during plant development.
|
To analyse in planta the effects of miR164-binding site mutation, we ethanol-induced from germination onwards the expression of the different CUC2 forms in the boundary domain in 15 randomly selected transgenic lines. Seedling development was normal for all lines tested until the formation of the first leaves. Scoring 10-day-old seedlings revealed that the expression of CUC2-m4 led to severe leaf growth inhibition with absence of any visible leaf in the most extreme case, or two smaller or unequal leaves in the milder cases (Fig. 3D,E). Expression of CUC2-m1 led also to retarded leaf development though in a smaller proportion of transgenic lines and with a milder effect (Fig. 3D,E). Expression of wild-type CUC2 or CUC2 mutated outside the miR164-binding site (CUC2-c1 and CUC2-c4) had no effect on leaf development (Fig. 3E).
We further investigated the developmental effects of the disrupted miR164-binding site by analysing STM::ALCR-alcA::erGFP control lines and STM::ALCR-alcA::erGFP alcA::CUC2-m4 lines that were ethanol-induced for 6 days just after bolting. No modifications of the mature flowers were observable during the first 3 weeks after induction. The mature flowers formed during the beginning of the fourth week were modified as petal number could be reduced while sepal spacing was increased (Fig. 3F).
In conclusion, these results showed that miR164 regulation of CUC2 was essential for plant development. In particular, disruption of the miR164-binding site in CUC2 and miR164 overexpression had opposite effects on sepal boundary formation.
The miR164-binding site is required for in planta regulation of CUC2 messengers
We compared CUC2 mRNA accumulation in STM::ALCR-alcA::erGFP
alcA::CUC2-wt and STM::ALCR-alcA::erGFP alcA::CUC2-m4
lines. Non-induced 9 day-old seedlings were ethanol-induced overnight, before
tissue sampling. This short induction allowed us to compare the direct effects
on CUC2 mRNA levels by reducing secondary effects resulting from the
modification of meristem organisation and thus of the size of the
STM-expressing domain. Variable expression levels were observed
between lines, but the average expression in CUC2-m4 lines was about
six times higher than in CUC2-wt lines
(Fig. 4A). This showed that
disruption of the miR164-binding site in CUC2 resulted in
higher mRNA accumulation levels. Accumulation of cleavage products was
reported for miRNA-mediated regulation of target mRNA levels
(Kasschau et al., 2003;
Llave et al., 2002
;
Palatnik et al., 2003
). We
could detect a cleavage product in the 2x35S::CUC2 but not in the
2x35S::CUC2-m4 lines (Fig.
4B). No such cleavage product of CUC2 could be detected
in wild-type, miR164 overexpressers or in the
STM::ALCR-alcA::erGFP alcA::CUC2 lines, possibly owing to low
representations of the CUC2 transcripts in these lines resulting from
their localised expression (result not shown).
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Discussion |
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By overproducing miR164, we showed that this miRNA reduced the
mRNA level of four out of the five predicted targets
(Rhoades et al., 2002). This
downregulation resulted from miRNA-guided cleavage of the mRNA, as we could
detect degradation products of the CUC2 mRNA that were dependent on
the presence of a miR164 complementary site in CUC2,
confirming previous identification of CUC1 and CUC2 mRNA
cleavage products (Kasschau et al.,
2003
). We did not observe a reduction of NAC1 mRNA level
in inflorescences of miR164 overexpressers. Several hypotheses could
account for this. NAC1 may not be a real target of miR164.
miR164-mediated degradation of NAC1 mRNA could also be
developmentally regulated and not occur under the conditions we tested.
Alternatively, miR164 may not regulate NAC1 activity via
mRNA cleavage but through translational attenuation as generally observed for
animal miRNA and for some plant miRNAs
(Aukerman and Sakai, 2003
;
Chen, 2004
). Could such a dual
mechanism for miR164 be the result of differences in the target
sequences? Two or three mismatches are observed between miR164 and
the five predicted targets (Rhoades et
al., 2002
). However, if pairings between G and U are allowed, two
mismatches subsist for NAC1-miR164 complexes, whereas only one exists
for the four targets for which cleavage is observed. It would therefore
suggest that the mode of action of miR164 depends on the extent of
its homology with the target, as observed for small RNAs in animals
(Doench et al., 2003
;
Zeng et al., 2003
). It must be
noted that, although G-U base pairing is possible, our mutational analysis of
the miR164-binding site of CUC2 (CUC2-m1) showed
that G-U pairing is not functionally equivalent to G-C pairing.
We show that during early phases of sepal boundary development, cell
proliferation is not repressed and that there is no strict restriction of cell
division orientations. Therefore, transverse cell division that can
potentially lead to boundary enlargement can occur unless the boundary
identity is rapidly switched off. We provide evidence that
miR164-dependent degradation of CUC1 and CUC2
transcripts constrains the expansion of the boundary domain resulting from
boundary cell proliferation. First, boundary enlargement was observed when a
miR164-resistant CUC2 form was expressed in the boundary
domain using STM regulatory sequences. Second, similar boundary
defects were observed in mutants with reduced miR164 levels. What is
the relation of miR164-dependant boundary size regulation with cell
proliferation? MiR164 may switch off the CUC1,2 function
after division in one of the daughter cells and thus induce different cell
identities. Alternatively, the link with cell division could be looser.
miR164 may switch off the CUC1,2 function in the outermost
boundary cell in response to boundary enlargement, resulting either from the
proliferation of this cell or from another cell. In both cases, a boundary
cell would reset its identity and adopt either a meristem or a primordium
identity. The latter could account for the earlier observation that, during
pea leaf development, cells are recruited into the growing primordium from
adjacent domains (Lyndon,
1970).
miRNAs are evolutionary conserved in both plants and animals. miR164
homologues have been reported for rice and tobacco
(Mallory et al., 2002;
Reinhart et al., 2002
), and
could be found in database for poplar and Medicago truncatula. In
addition to Arabidopsis, a potential miR164-binding site is
present in NAC genes of rice
(Rhoades et al., 2002
),
petunia, Antirrhinum majus, soybean and bean. At least two of them,
NAM and CUP, have a similar role to the Arabidopsis CUC
genes in petunia and Antirrhinum, respectively
(Souer et al., 1996
;
Weir et al., 2004
). Therefore,
the mechanism of boundary stabilisation we described for Arabidopsis
is likely to be evolutionary conserved. A similar conservation of the
miRNA-target function has been described for the control of leaf polarity
between Arabidopsis and maize
(Floyd and Bowman, 2004
;
Juarez et al., 2004
;
Kidner and Martienssen,
2004
).
We have shown that CUC1 and CUC2 mRNAs are targeted for
degradation by miR164, whereas CUC3 is not directly
regulated by the miRNA. Why is CUC3 not a target of miR164?
A higher level of redundancy seems to exist within the CUC genes in
Arabidopsis than in other species
(Souer et al., 1996;
Weir et al., 2004
).
Nevertheless, although the CUC1, CUC2 and CUC3 genes have
all a role in boundary specification, their contribution is not identical.
First, genetic analyses suggest that the contribution of CUC3 to
cotyledonary boundaries is more important than those of CUC1 and
CUC2 (Vroemen et al.,
2003
). Second, expression patterns of the CUC1, CUC2 and
CUC3 genes differ slightly during embryogenesis
(Vroemen et al., 2003
).
Finally, the CUC2 expression domain is reduced in the embryo of the
pin-formed1 mutant, whereas the CUC1 domain is expanded
(Aida et al., 2002
), suggesting
that these two genes differ in their response to the signalling molecule auxin
involved in primordia patterning
(Reinhardt et al., 2003
). It
appears therefore that the precise regulation of CUC1, CUC2 and
CUC3 involves different mechanisms. In this context, miRNA-regulation
is apparently an additional level of control. Besides, it is possible that
another, not yet identified, miRNA could regulate CUC3.
Note added in proof
While this paper was under review, Mallory et al.
(Mallory et al., 2004)
described partially overlapping results.
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ACKNOWLEDGMENTS |
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Footnotes |
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