A MicroRNA as a Translational Repressor of

APETALA2 in Arabidopsis Flower Development

Xuemei Chen, et al.
Science 303, 2022 (2004);
DOI: 10.1126/science.1088060

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 REPORTS
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recipient), and 53 (6 donor and 3 recipient). performed in the UAB CFAR Central Virus Core (P30-
AI-27767). This work was supported by NIH grants
AI-51231 (E.H.), AI-40951 (S.A.), U01-AI-41530 (G.S.
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A MicroRNA as a Translational and a novel protein, HEN1 [reviewed in (3)].

The two founding members of Caenorhabdi-

Repressor of APETALA2 in tis elegans miRNAs, lin-4 and let-7, inhibit

the translation of their target mRNAs through
imperfect base-pairing interactions with their
Arabidopsis Flower Development targets (4, 5). Arabidopsis miRNAs show a
higher degree of sequence complementarity
Xuemei Chen to their potential targets, and, like short inter-
fering RNAs, several plant miRNAs direct
Plant microRNAs (miRNAs) show a high degree of sequence complementarity to, the cleavage of their target RNAs (6–9).
and are believed to guide the cleavage of, their target messenger RNAs. Here, I show Here, I report the role of an Arabidopsis
that miRNA172, which can base-pair with the messenger RNA of a floral homeotic miRNA, miRNA172, in the control of floral
gene, APETALA2, regulates APETALA2 expression primarily through translational organ identity and floral stem cell prolifera-
inhibition. Elevated miRNA172 accumulation results in floral organ identity defects tion as a potential translational repressor of a
similar to those in loss-of-function apetala2 mutants. Elevated levels of mutant floral homeotic gene.
APETALA2 RNA with disrupted miRNA172 base pairing, but not wild-type Four organ types are specified in the floral
APETALA2 RNA, result in elevated levels of APETALA2 protein and severe floral meristem by the combinatorial actions of
patterning defects. Therefore, miRNA172 likely acts in cell-fate specification as three classes of transcription factors, the flo-
a translational repressor of APETALA2 in Arabidopsis flower development. ral A, B, and C genes [reviewed in (10)].
APETALA2 (AP2, a class A gene) and
MicroRNAs (miRNAs), ⬃22-nucleotide non- transcripts by the enzyme Dicer [reviewed in
coding RNAs that regulate protein-coding (1, 2)]. In Arabidopsis, the accumulation of Waksman Institute, Rutgers University, Piscataway,
RNAs, are processed from longer hairpin miRNAs requires a Dicer homolog, DCL1, NJ 08854, USA. E-mail: xuemei@waksman.rutgers.edu

2022 26 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org


AGAMOUS (AG, a class C gene) specify the
identities of the perianth and reproductive
organs, respectively, and act antagonistically
to restrict each other’s activities to their prop-
er domains of action within the floral meris-
tem. We have identified HEN1 as a gene re-
quired for class C activity in the flower, because
recessive hen1 mutations result in reproductive-
to-perianth organ transformation in the hua1-1
hua2-1 background, which is partially compro-
mised in class C activity (11, 12). The require-
ment of HEN1 for the accumulation of
miRNAs (13) suggests that the absence of a
miRNA(s) is responsible for the floral homeotic
transformation in hua1-1 hua2-1 hen1 flowers.
MiRNA172, a miRNA highly comple-
mentary to a region in the mRNAs of AP2
(14) (Fig. 1A), At2g28550, At5g60120, and
At5g67180, which encode proteins with AP2
domains, is a candidate miRNA in flower
development. The putative miRNA172 bind-
ing sites are located within the coding regions
of the genes but are outside of the conserved
AP2 domains. To test whether AP2 is regu-
lated by miRNA172, I first studied AP2 ex-
pression in genotypes in which miRNA172
accumulates to low levels, such as hen1 and
dcl1 mutants (Fig. 1B). AP2 RNA abundance
in these genotypes was similar to that in wild
type (Fig. 1C) (9). AP2 protein amounts in
hen1 and dcl1 inflorescences, however, were
about 2 to 3 times that in wild type (Fig. 1D),
suggesting that AP2 is regulated at the trans-
lational or posttranslational levels by a
miRNA(s) absent in hen1 and dcl1 plants.
Elevated AP2 accumulation is likely the
cause of the homeotic transformation in
hua1-1 hua2-1 hen1-1 flowers, because a
severe loss-of-function ap2 allele, ap2-2 (15),
rescues the hua1-1 hua2-1 hen1-1 floral ho-
meotic phenotypes (12).
To determine whether AP2 is regulated by
miRNA172, I tested the effect of elevated
miRNA172 level on AP2 expression in planta.
Five genes in the Arabidopsis genome (table
S1) can give rise to three miRNA172 species
with one nucleotide difference. All three
miRNA172 species can base-pair with AP2
RNA with no or one mismatch (Fig. 1A). Each
of four MIR172 genes, MIR172a-1, a-2, b-2,
and c (13), was fused to two copies of the
cauliflower mosaic virus 35S enhancer, which
drives high-level and near ubiquitous expres-
sion, and was introduced into wild-type plants.
Most T1 transgenic lines containing each
MIR172 gene but not MIR173 (an unrelated
miRNA gene) or the vector alone displayed
accelerated floral transition (Fig. 2A). In addi-
tion, plants harboring 35S::MIR172a-1, a-2, or
b-2, but not 35S::MIR172c, the vector DNA, or
35S::MIR173, showed floral homeotic pheno-
types similar to those of ap2 loss-of-function
mutants, such as ap2-2 or ap2-9 (15) (Figs. 2B
and 3, B and C). Similar phenotypes were also
observed in an activation-tagging line that over-
www.sciencemag.org SCIENCE VOL 303 26 MARCH 2004
REPORTS
expressed miRNA172a-2 (16). Some cauline miRNA172 (Fig. 2, C to E). Because the
leaves of 35S::MIR172a-1, 35S::MIR172a-2, abundance of miRNA172 in wild-type plants
and 35S::MIR172b-2 lines had stigmatic tissues is low in young seedlings and high in inflo-
(Fig. 3D), suggesting elevated AG expression. rescences (13), the increase in miRNA172
Indeed, AG RNA accumulation was elevated in abundance in 35S::MIR172 lines was more
the cauline leaves of 35S::MIR172a-1 plants evident in young seedlings than in inflores-
relative to control transgenic plants (fig. S1). cences (Fig. 2, C and E). Despite the presence
Some 35S::MIR172a-1 and a-2 plants also had of ap2-like phenotypes in these lines, AP2
rosette leaves that curled upward (Fig. 3E), a RNA accumulation was similar to that in
phenotype found in plants expressing AG in control transgenic plants (Fig. 2F). AP2 pro-
leaves (17, 18). Because severe loss-of-function tein, although readily detectable in wild type,
ap2 mutants do not have curly leaves or was undetectable in 35S::MIR172a-1 inflo-
carpelloid leaves, the leaf phenotypes of rescences (Fig. 1D). This indicates that
35S::MIR172 lines indicate that miRNA172 miRNA172 regulates AP2 expression at the
regulates, in addition to AP2, other genes that translational level.
repress AG in leaves. Expression of MIR172 with the 35S en-
RNA filter hybridization showed that hancer demonstrated the ability of MIR172 to
these transgenic lines had higher levels of regulate AP2 expression in planta when it is
Downloaded from www.sciencemag.org on December 30, 2008
Fig. 1. Sequence comple-
mentarity between AP2 RNA
and miRNA172 and AP2
RNA and protein accumula-
tion in various genotypes.
(A) A diagram of the AP2
mRNA showing the AP2 do-
mains (hatched rectangles)
and the miRNA172 binding
site (black rectangle). The
mutant nucleotides in AP2m1
and AP2m3 are circled. Although AP2m1 causes a Phe-to-Leu amino acid substitution (underlined
codon), AP2m3 does not change the amino acid sequence. Nucleotides in miRNA172 that can
base-pair with AP2 RNA (with G:U pairing allowed) are in bold. The ap2-2 allele is a splice acceptor
site mutation that would generate a stop codon at the indicated position (arrowhead). 3⬘UTR, 3⬘
untranslated region; 5⬘UTR, 5⬘ untranslated region. (B) Accumulation of miRNA172 in inflores-
cences. The stained gel below indicates the amount of RNA in each lane. wt, wild type. (C) AP2 RNA
abundance in inflorescences. The numbers indicate the relative abundance of AP2 RNA in various
genotypes. (D) AP2 protein abundance in inflorescences. Although the AP2 antisera reacted with a
number of proteins (one indicated by the asterisk), the identity of the AP2 signal was revealed by
its absence in the ap2-2 mutant. The numbers indicate the abundance of AP2, as calculated by
using phosphoenolpyruvate carboxylase (PEPC) as the loading control.
2023
 REPORTS
expressed from an exogenous promoter. Is
miRNA172 normally present at the expected
time during flower development? A modified
in situ hybridization procedure showed that
the antisense miRNA172 probe gave strong
signals in stage 1 floral primordia (Fig. 3F).
The signal persisted in all four floral whorls
until stage 7, when miRNA172 appeared to
be concentrated in the inner two floral whorls
(Fig. 3H). The signal was unlikely to be a
result of the miRNA precursor (fig. S2). The
sense probe did not give any hybridization
signals (Fig. 3, G and I). The presence of
miRNA172 in developing flowers indicates
that miRNA172 can regulate AP2 in floral
patterning. In fact, the later accumulation of
miRNA172 in the inner two whorls may
cause AP2 protein to be concentrated in the

Downloaded from www.sciencemag.org on December 30, 2008

outer two floral whorls, where AP2 acts to
specify perianth identities.
The ability of miRNA172 to repress AP2
expression in vivo and the presence of
miRNA172 in the flower suggest that
miRNA172 normally keeps AP2 expression
level low during flower development. To as-
sess the importance of this regulation in flow-
er development and to determine whether this
regulation is direct, I introduced wild-type
AP2 cDNA or AP2m1, a mutant AP2 cDNA
with six mismatches to miRNA172 (Fig. 1A),
into wild-type plants and expressed them un-
der the control of a 35S promoter. As a result,
100 and 82 independent transgenic plants for
35S::AP2 and 35S::AP2m1, respectively,
were obtained. Dramatic differences were
found in the severity and frequency of floral
defects between the two transgenic T1 popu-
lations (Fig. 4A). About 60% of 35S::AP2m1
plants had flowers with an enlarged floral
meristem surrounded by many whorls of sta-
minoid organs or petals (Fig. 3, J and K).
Some floral defects, such as the loss of floral
determinacy and the transformation of sta-
mens to petals, resemble those in ag severe
loss-of-function mutants, which is consistent
with the ability of AP2 to negatively regulate
AG. Other floral defects, such as the enlarge-
ment of the floral meristem and the extreme
excess of stamens, suggest that AP2 regulates
other floral patterning genes. Alternatively,
these phenotypes may have resulted from
elevated AP2 expression and do not represent
the natural functions of AP2. In contrast to
35S::AP2m1 plants, the majority of 35S::AP2
plants had normal flowers (Fig. 4A). Only a
small number of 35S::AP2 plants showed
mild floral defects (Fig. 3L). The 35S::AP2
plants with normal flowers had elevated AP2
RNA accumulation comparable to those in
35S::AP2m1 plants with severe floral defects
(Fig. 4C and fig. S3). However, AP2 protein
abundance was elevated in 35S::AP2m1 but
not 35S::AP2 plants (Fig. 1D). The similarity
in AP2 RNA abundance but difference in
AP2 protein levels indicates that miRNA172
20-day seedlings (the five lanes on the left) and in inflorescences (the two lanes on the right) of
T2 lines.
regulates AP2 expression at the translational
level. Note that AP2 protein abundance in
35S::AP2m1 lines is similar to that in hen1 or
dcl1 plants (Fig. 1D), although the hen1 or
dcl1 floral defects are much weaker than
those of 35S::AP2m1 plants. This may be a
result of reduced accumulation of many miR-
NAs, some of which may regulate targets that
act antagonistically to AP2, in hen1 or dcl1
plants. Alternatively, some 35S::AP2m1 phe-
notypes may be a result of the exogenous
promoter used in the study.
Many 35S::AP2 and 35S::AP2m1 plants
also exhibited delayed floral transition (Fig.
4B). Interestingly, about a quarter of
35S::AP2 plants, although not exhibiting any
floral phenotypes, were later flowering than
35S::AP2m1 plants, including those with se-
vere floral defects (Fig. 4B). Although AP2
does not appear to function in floral transi-
tion, two closely related genes, At2g28550
(TOE1) and At5g60120 (TOE2), which also
have a miRNA172 binding site, act as repres-
Fig. 2. Analyses of 35S::MIR172 transgenic
lines. (A) Leaf numbers of T1 transgenic lines.
(B) Percentage of T1 transgenic lines with phe-
notypes that resemble plants with ectopic AG
expression, such as curly leaves, carpelloid
leaves, and ap2-like flowers. The total number
of independent lines analyzed in (A) and (B) is
in parentheses. The miRNA172 abundance in
(C) 20-day seedlings of T2 lines, (D) 30-day T1
lines, and (E) inflorescences of T2 lines. The
stained gels in (C) and (E) and the 5S rRNA
hybridization in (D) indicate the amount of
total RNA used. (F) AP2 RNA accumulation in
sors of flowering (16). Given that 35S::AP2
and 35S::AP2m1 lines with moderately or
severely delayed floral transition showed
high levels of AP2 RNA (Fig. 4C and fig. S3)
but only 35S::AP2m1 lines had high levels of
AP2 protein (Fig. 1D), it is likely that the
late-flowering phenotypes in 35S::AP2 and
35S::AP2m1 plants were caused by two dis-
tinct mechanisms. The increased abundance
of AP2 protein in 35S::AP2m1 may allow
AP2 to act through the TOE pathway to delay
floral transition. On the other hand, the in-
creased abundance of AP2 RNA in 35S::AP2
lines may compete with the TOE RNAs for
miRNA172 regulation such that more TOE
proteins accumulate and delay floral transi-
tion. Consistent with this hypothesis,
35S::MIR172a-1 rescues the late-flowering
phenotype of 35S::AP2 plants (19).
In addition to disrupting the miRNA172
binding site, the AP2m1 mutation also intro-
duced an amino acid substitution (Phe to Leu,
Fig. 1). To evaluate whether the phenotypes

2024 26 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org

 REPORTS

Fig. 3. Phenotypes of 35S::MIR172, 35S::AP2, and 35S::AP2m1 lines floral meristems (FM) [(F) and (G)] and stage 7 flowers [(H) and (I)].

Downloaded from www.sciencemag.org on December 30, 2008

and in situ localization of miRNA172. (A) A wild-type flower. (B) An
ap2-9 flower with first-whorl organs transformed into carpels (arrow).
(C) A 35S::MIR172a-1 flower that closely resembles ap2 flowers. A
cauline leaf with stigmatic tissues (D, arrows) and a curly rosette leaf
(E) from 35S::MIR172a-1 plants. In situ hybridization using an anti-
sense [(F) and (H)] or sense [(G) and (I)] miRNA172 probe on
longitudinal sections of inflorescence meristems (IM) with two young
Hybridization signals are represented by the blue color. Size bar, 50
␮m. The numbers in (H) and (I) represent the positions of the organs,
with 1 being the outermost whorl. (J) A 35S::AP2m1 flower with
numerous petals and loss of floral determinacy. (K) A 35S::AP2m1
flower with many staminoid organs and an enlarged floral meristem
(arrow). (L) A 35S::AP2 flower with more stamens than the
wild type.

miRNAs can regulate their target mRNAs

through one or both mechanisms, translational
inhibition and transcript cleavage.

References and Notes

Fig. 4. Analyses of 35S::AP2 and 35S::AP2m1 trans-
genic T1 lines. (A) The frequency of floral defects in
35S::AP2 and 35S::AP2m1 lines. (B) Flowering time
as represented by the numbers of total leaves pro-
duced before flowering or the numbers of days it
takes for the stem to reach 0.5 cm in length. (C) AP2
RNA accumulation in inflorescences from T1 trans-
genic plants containing the vector alone, 35S::AP2
(with normal flowers but flowers extremely late), or
35S::AP2m1 (with severe floral defects and flowers
moderately late). Note that the tissues used were
the same as the ones used in AP2 protein analysis
shown in Fig. 1D. Numbers at the bottom indicate
the relative amount of AP2 RNA in the various
genotypes.
of 35S::AP2m1 plants were caused by the
amino acid change, I generated 35S::AP2m3
plants, which harbor a truly silent mutation in
the miRNA172 binding site (Fig. 1). Analysis
of 88 independent transgenic plants showed
that 35S::AP2m3 had nearly identical pheno-
types to those of 35S::AP2m1 (fig. S4).
During flower development, miRNA172 re-
presses the expression of AP2. This regulation
is likely crucial for the proper development of
the reproductive organs and for the timely ter-
mination of floral stem cells. Furthermore, this
regulation appears to be mediated by direct
sequence complementarity between AP2
mRNA and miRNA172 and occurs primarily at
the level of translation. MiRNA172-guided
cleavage of AP2 mRNA may also occur at a
low level, because potential cleavage products,
presumably mediated by miRNA172, have
been previously detected by rapid amplification
of cDNA ends–polymerase chain reaction (9,
15). This and other studies show that plant
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20. I thank B. Krizek for the AP2 cDNA clone and for
communicating unpublished results; T. Ito for the
pMAT137 vector; J. Liu and W. Park for technical
assistance; A. Singson for providing equipment; Z. Liu,
D. Wagner, S. Poethig, and R. Kerstetter for helpful
discussions; R. Kerstetter and R. Steward for com-
ments on the manuscript; and NIH (R01 GM61146)
for financial support.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1088060/DC1
Materials and Methods
Figs. S1 to S4
Table S1
16 June 2003; accepted 18 July 2003
Published online 31 July 2003;
10.1126/science.1088060
Include this information when citing this paper.

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