Download Inflorescence Meristem Identity in Rice Is Specified

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Inflorescence Meristem Identity in Rice Is Specified by
Overlapping Functions of Three AP1/FUL-Like MADS Box
Genes and PAP2, a SEPALLATA MADS Box Gene
C W
Kaoru Kobayashi,a Naoko Yasuno,a Yutaka Sato,b Masahiro Yoda,a Ryo Yamazaki,a Mayumi Kimizu,c
Hitoshi Yoshida,d Yoshiaki Nagamura,b and Junko Kyozukaa,1
a Graduate
School of Agriculture and Life Sciences, University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan
c Crop Development Division, National Agricultural Research Center, National Agriculture and Food Research Organization,
Jo-etsu, Niigata 943-0193, Japan
d Rice Research Division, National Institute of Crop Science, National Agriculture and Food Research Organization,
Tsukuba 305-8518, Japan
b National
In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of
the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser
microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/
FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice
(Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of
these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of
PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple
knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots
were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the
precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1
mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to
specify the identity of the IM downstream of the florigen signal.
INTRODUCTION
Plants progress through distinct developmental phases in their
life cycles (reviewed in Huijser and Schmid, 2011). The transition
from vegetative to reproductive growth, generally called flowering, is vital for reproductive success. Flowering occurs in response to environmental and developmental signals, such as
daylength, plant age, and temperature (reviewed in Liu et al.,
2009; Wellmer and Riechmann, 2010). Multiple flowering signals
are eventually transmitted to the shoot apical meristem (SAM),
a group of stem cells in the shoot apex, where they are converted to local cues that evoke the developmental phase
transition. In Arabidopsis thaliana, SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and FLOWERING LOCUS T (FT)
function as the main floral integrators that transmit multiple
flowering signals to the meristem. SOC1 is one of the first genes
to be induced in the SAM at this transition (Lee et al., 2000). FT is
1 Address
correspondence to [email protected].
The author 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) is: Junko Kyozuka
([email protected]).
C
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www.plantcell.org/cgi/doi/10.1105/tpc.112.097105
a major component of florigen, a long-distance flowering signal
(Abe et al., 2005; Wigge et al., 2005). The FT protein is produced
in leaves and then transported to the SAM, where it interacts
with FLOWERING LOCUS D (FD). In the SAM, the SOC1 and FTFD complex promote expression of several downstream genes,
including LEAFY, APETALA1 (AP1), and FRUITFULL (FUL),
which specify floral meristem identity (Abe et al., 2005; Wigge
et al., 2005; Corbesier et al., 2007).
The aerial part of a plant is generated by the SAM, which
produces leaves during vegetative development. Upon transition to reproductive development, the SAM converts into an
inflorescence meristem (IM) and starts to generate components
of the inflorescence. Development of the IM is variable depending on species, leading to formation of species-specific
inflorescence architecture. The conversion from the vegetative
SAM to the IM is less well understood at the molecular level than
events such as flowering time control, floral meristem specification, and flower development. Several genes show a dramatic
change in their expression patterns in the meristem at phase
transition (Cardon et al., 1997; Hempel et al., 1997; Lee et al.,
2000; Samach et al., 2000; Torti et al., 2012). This reflects
a global change in meristem gene expression that is associated
with the establishment of IM identity (Schmid et al., 2003; Park
et al., 2012).
In grasses, the shift of the meristem phase from the SAM
to the IM is accompanied by clear changes in a number of
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developmental and morphological traits. In rice (Oryza sativa),
the first morphological change is a rapid increase in meristem
size (Hoshikawa, 1989). The IM starts to initiate inflorescence
branch meristem (BM) primordia. Heading date 3a (Hd3a) and
RICE FLOWERING LOCUS T1 (RFT1) function as major components of florigen in rice (Tamaki et al., 2007; Komiya et al.,
2008, 2009). Recent findings show that Hd3a protein generated
in leaves is transported to the cytoplasm of shoot apex cells
where it interacts with 14-3-3 proteins (Taoka et al., 2011). The
Hd3a/14-3-3 complex then enters the nucleus and activates the
rice ortholog of FD through phosphorylation. The absence of
both Hd3a and RFT1 functions completely blocks transition to
reproductive growth (Komiya et al., 2009). This implies that, in
the meristem, FD and possibly other downstream targets of
Hd3a and RFT1 induce genes that promote meristem phase
transition. Expression of both MADS14 and MADS15, which
encode AP1/FUL-like MADS domain proteins, was suppressed
in Hd3a and RFT1 double knockdown plants, suggesting that
these genes function downstream of the florigen signal (Komiya
et al., 2008, 2009). Moreover, it has been demonstrated that
MADS15 transcription is induced by phosphorylated FD in the
presence of Hd3a (Taoka et al., 2011). These reports suggest
that MADS14 and MADS15 may be regulators of phase transition in the meristem; however, neither their functions nor their
expression patterns in the meristem at phase transition have
been demonstrated.
In this study, we demonstrate that four MADS box genes,
PANICLE PHYTOMER2 (PAP2) (Kobayashi et al., 2010), MADS14,
MADS15, and MADS18 are activated in the meristem at phase
transition and function redundantly in specifying IM identity in rice.
RESULTS
PAP2 Expression Is Induced in the Meristem upon
Reproductive Transition
Rice PAP2 encodes MADS34, a grass-specific SEPALLATA
(SEP) subfamily MADS domain protein (see Supplemental Figure
1 online). PAP2 plays multiple roles, including the determination
of spikelet meristem (SM) identity and the suppression of the
extra growth of glumes (Gao et al., 2010; Kobayashi et al., 2010).
To understand PAP2 function further, we analyzed its spatial
and temporal expression throughout inflorescence development. A 2.2-kb fragment of the PAP2 promoter region was
fused with the b-glucuronidase (GUS) gene and transformed into
wild-type rice plants. T1 seedlings were grown for 7 d under
long-day (LD) conditions and then transferred to short-day (SD)
conditions, which promote the transition to the reproductive
phase. GUS activity was not detected in the SAM during the
vegetative phase (Figures 1A and 1B). Upon transition to reproductive development, clear induction of PAP2:GUS activity
throughout the SAM was observed (Figure 1C). GUS activity
became localized to the incipient SM at the spikelet initiation
stage (Figures 1D and 1E). During spikelet organ development,
GUS activity was observed in initiating glumes (Figure 1F). PAP2
expression in the SM and glumes was consistent with the known
functions of PAP2, namely, promotion of spikelet identity and
Figure 1. Spatial and Temporal Distribution of PAP2 Promoter Activity.
(A) to (F) GUS staining in the shoot apex of PAP2:GUS plants at the
vegetative stage (A), slightly before reproductive transition (B), at the
transition stage (C), and at the spikelet initiation stage ([D] and [E]). A
close-up view of the region enclosed with a square in (D) is shown in (E).
GUS activity in the developing spikelet is shown in (F).
(G) and (H) In situ hybridization analysis of PAP2 expression in the shoot
apex at the transition stage (G) and in the inflorescence at spikelet initiation stage. Arrowhead, SAM; BM, branch meristem; SM, spikelet
meristem; FM, floral meristem.
(I) The relationship between meristem size and GUS activity is shown.
suppression of extra growth of glumes (Kobayashi et al., 2010).
Localization of PAP2 mRNA in the meristem at transition stage
and the SMs was confirmed by in situ hybridization analysis
(Figures 1G and 1H). To determine the timing of PAP2 induction
precisely, the relationship between the meristem size and the
onset of PAP2:GUS activity was examined (Figure 1I). Elongation of the SAM, a sign of the transition to reproductive growth,
was accompanied by a dramatic increase in GUS activity. These
results suggest that PAP2 is involved in reproductive transition
and/or the establishment of the identity of the IM.
Inflorescence Meristem Identity of Rice
A null allele of the PAP2 locus, pap2-1, does not show any
significant changes in the very early stages of inflorescence
development (Kobayashi et al., 2010). Because the SEP MADS
domain proteins usually function in complexes with other, related MADS domain proteins (reviewed in Gramzow and
Theissen, 2010), we postulated that the effects of the loss of
PAP2 activity during reproductive phase transition may be
masked by redundancy with other gene(s). To test this idea, we
searched for MADS box genes that show overlapping expression patterns with PAP2 in the SAM during the reproductive
transition. For this purpose, we adopted a laser microdissection
(LM) and microarray technique and specifically sampled SAMs
at various developmental stages (Figure 2A). All samples were
collected from plants grown in the field in 2008. SAMs from the
Vegetative 1 (V1) stage were harvested on June 24th before the
induction of RFT1, a primary determinant of rice flowering under
natural LD conditions (Komiya et al., 2009). V2 SAMs were
sampled on July 8th, several days after RFT1 induction was first
observed in the leaves. Because slight variations in size were
observed in the SAMs collected on July 15th, each SAM was
identified as being at either the transition (V/R) or elongation (R1)
stage, depending on its size as measured under the microscope.
In situ hybridization analysis of ABERRANT PANICLE ORGANIZATION1 (APO1) expression in V/R stage SAMs revealed a mixture
of APO1-expressing and nonexpressing SAMs (see Supplemental
Figure 2 online). Because induction of APO1 is concurrent with
reproductive transition (Ikeda-Kawakatsu et al., 2009), this indicates
that the V/R stage is a critical time in the transition of the SAM to
the IM. Inflorescences at the primary branch initiation, secondary
branch initiation, and spikelet initiation stages were collected as R2,
R3, and R4 stages (Figures 2A and 2B).
Microarray analysis was performed with RNAs isolated from
dissected meristems and very young inflorescences. The overall
profile of gene expression during meristem phase transition is
shown in Supplemental Figure 3 online. The levels of PAP2 RNA
increased from the V2 to the V/R stage, and this was consistent
with changes in PAP2:GUS activity (Figures 2B and 2C). In addition, expression of three MADS box genes (out of 34 MADS box
genes on the microarray) showed increases in expression between the V1 and the V/R stages (Figures 2B and 2C). Intriguingly,
these three genes are MADS14, MADS15, and MADS18, which
belong to the AP1/FUL-like subfamily of MADS box genes (Litt
and Irish 2003; Fornara et al., 2004; Preston and Kellogg, 2006;
see Supplemental Figure 1 online). The only other member of this
subfamily in rice, MADS20, showed no detectable expression in
meristems at any of the stages examined in this study. Two of the
three MADS box genes, MADS14 and MADS18, displayed similar
expression patterns in which the mRNA level increased between
the V1 and V2 stages (Figure 2C). Accumulation of MADS14
mRNA further increased from V2 to V/R, whereas the expression
of MADS18 remained relatively constant after the V2 stage.
MADS15 showed an expression pattern similar to PAP2; that is,
its mRNA level increased from the V2 to the V/R stage, accompanying the transition to reproduction.
The SEP subfamily of rice consists of five genes, including
PAP2 (Malcomber and Kellogg, 2005; Zahn et al., 2005). As
previously reported (Arora et al., 2007; Kobayashi et al., 2010),
none of the other four SEP genes, MADS1, MADS5, MADS7,
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Figure 2. LM Analysis of Genes Expressed in Shoot Meristems during
Reproductive Transition.
(A) Micrographs of the meristem stages that were analyzed. V1 and V2
are meristems collected before transition. V/R and R1 are meristems at
the transition stage and elongation stage, respectively. R2 and R3 are the
primary branch initiation and secondary branch initiation stages of the
inflorescences, respectively.
(B) Heat map representing the expression of rice MADS box genes during
early stages of inflorescence development. Genes that show a significant
level of expression in at least one of the 24 arrays performed are included.
The intensity of the red color represents absolute expression of each gene.
(C) Expression patterns of four MADS box genes that showed a significant increase in expression level at reproductive transition. The values
represent mean expression levels 6 SD (n = 3 to 5) obtained from the
microarray analysis of microdissected meristems.
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and MADS8, showed detectable expression at the early stages
of inflorescence development studied here except that a low
level of MADS5 expression was observed in the spikelet initiation stage (R4) (Figure 2B).
Inflorescence Development Was Severely Perturbed in the
MADS Quadruple Knockdown Plants
To understand the role of PAP2 and the three AP1/FUL-like
genes during reproductive transition and early stages of inflorescence development, we generated transgenic rice plants in
which the expression levels of MADS14, MADS15, or MADS18
were reduced by RNA interference (RNAi). Despite a significant
reduction in the levels of mRNA from each of these genes,
neither inflorescence development nor flowering time was altered in single RNAi plants (see Supplemental Figure 4 online;
Fornara et al., 2004). We then produced pMADS14;15;18i, an
RNAi construct that simultaneously suppresses expression of
MADS14, MADS15, and MADS18. This construct was introduced into wild-type, pap2-1/+, and pap2-1 plants. Plants
transformed with the empty RNAi vector were used as controls.
Specificity of the RNAi target for the three genes was confirmed
by quantitative real-time RT-PCR (see Supplemental Figure 5
online). The expression level of MADS20, another member of the
AP1/FUL-like genes of rice, was not clearly affected in transgenic lines containing pMADS14;15;18i.
Growth of all transgenic plants was normal during the vegetative phase, whereas inflorescence development was severely
perturbed in the homozygous mutant pap2-1 plants transformed
with pMADS14;15;18i (Figure 3). In normal rice development,
reproductive transition is accompanied by the initiation of stem
elongation. The stem gradually elongates during the early stages
of inflorescence development, with rapid elongation starting
a few days before heading. This results in the exposure of the
inflorescence as the flowers open (Figures 3A and 3B). Although
extensively delayed, stem elongation finally occurred and proceeded very slowly in the MADS14;15;18i pap2-1 quadruple
knockdown lines, indicating that reproductive transition began
in these plants. However, leaf initiation continued even after the
start of stem elongation (Figure 3C). The leaves produced during
the later stages showed various abnormalities, including serration and altered phyllotaxy (Figure 3C).
The severity of defects in inflorescence development varied
among transgenic lines. In weakly affected lines, although
delayed and reduced in size, the inflorescence was eventually
formed. In moderate lines, the vegetative SAM was observed in
the growing inflorescence, indicating incomplete reproductive
transition (see Supplemental Figure 6 online). In strong lines, the
inflorescence was not formed (see below). The severity roughly
correlates with the effectiveness of knockdown of the three AP1/
FUL-like genes (see Supplemental Figure 5 online). We chose
two severe quadruple knockdown lines (q8 and q10) and analyzed their reproductive development by scanning electron microscopy (Figures 4A to 4J), sectioning (Figures 4K to 4R), in situ
hybridization (Figures 4S and 4T), and observation under the
microscope (Figures 4U to 4Z). In wild-type rice, primary inflorescence branches arise in the axils of bracts (extremely reduced leaves) in a spiral phyllotaxy (Figures 4A to 4C, 4L, 4U,
Figure 3. Inflorescence Development of MADS14;15;18i/pap2-1 Quadruple Knockdown Plants.
(A) Nontransgenic wild-type (left) and MADS14;15;18i pap2-1 quadruple
knockdown (right) plants.
(B) Inflorescences on a wild-type plant after heading.
(C) Heading never occurred in the quadruple knockdown lines. Instead,
leaf production continued after heading had occurred in the wild-type
plants. A close-up view of the youngest leaf showing a serration (arrowhead) is shown in the inset.
[See online article for color version of this figure.]
and 4V). Each primary branch forms secondary branches and
spikelets (Figures 4D, 4E, 4M, and 4N). Hair-like structures arise
on the bracts (Figure 4C), and the developing inflorescence is
covered with the hair-like structure by the spikelet initiation
stage (Figures 4D, 4E, and 4V). Change to spiral phyllotaxy was
observed in both q8 and q10, indicating the start of reproductive
transition and consistent with the start of stem elongation (Figures 4F to 4I). However, unlike wild-type inflorescence development in which primary branches are formed, initiation of
vegetative leaf primordia continued (Figures 4G to 4J, 4P, 4Q,
and 4R). Axillary meristems were formed in the axils of these
leaves and grew as shoots (Figures 4G, 4H, and 4W). This
process was reiterated and led to the formation of multiple shoots
on one stem (Figures 4I, 4J, 4Q, 4X, 4Y, and 4Z). Spikelets were
produced in the wild-type inflorescence (Figures 4M and 4N),
whereas shoot formation was repeated and each meristem continued to generate leaf primordia in the quadruple knockdown
plants (Figures 4Q and 4R).
To determine further the identity of meristems in the quadruple
knockdown plants, we performed in situ hybridization analysis of
APO1, which is normally expressed in the IM and the BM but not in
the vegetative SAM (Ikeda-Kawakatsu et al., 2009). Absence of the
APO1 signal in the quadruple knockdown plants confirmed that the
axillary meristems retain the characteristics of the vegetative SAM
(Figures 4S and 4T). Our combined data suggest that progression
to reproductive transition was severely blocked in MADS14;15;18i
pap2-1 plants; thus, IM identity was not established. We propose
that PAP2 and three AP1/FUL-like genes act redundantly to promote IM identity and are thus IM identity genes.
AP1/FUL-Like MADS Box Genes Are Involved in the
Regulation of Flowering
In contrast with the severe defects observed in the quadruple
knockdown plants, few abnormalities was observed in inflorescence development of the MADS14;15;18i wild-type and
Inflorescence Meristem Identity of Rice
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Figure 4. Impaired Inflorescence Development in MADS14;15;18i pap2-1 Quadruple Knockdown Plants.
(A) to (J) Scanning electron microscopy analysis of wild-type ([A] to [E]) and MADS14;15;18i pap2-1 ([F] to [J]) apices. Each shoot and leaf like
structure are indicated with an arrow and yellow dot, respectively ([F] and [G]).
(K) to (N) Sections of wild-type inflorescences immediately after transition (K), at the inflorescence branch initiation stage (L), and at the spikelet
initiation stage (M). A close-up view of a developing spikelet from (M) is shown in (N).
(O) to (R) Sectioning of an MADS14;15;18i pap2-1 apex. Shoot apexes at an early stage (O) and the later stage ([P] to [Q]) are shown. The apical
meristem continued to initiate leaf primordia. Multiple shoots derived from axillary meristems of the leaves are observed (Q), and each meristem
appears as a vegetative SAM producing leaf primordia. Each shoot is indicated with a star ([P] and [Q]). A close-up view of a meristem initiating leaf
primordia in (Q) is shown in (R).
(S) and (T) In situ hybridization showing APO1 expression (dark stain) in a developing inflorescence at the secondary branch initiation stage in the wildtype apex (S). APO1 expression is not observed in the apex of the MADS14;15;18i pap2 plant (T).
(U) and (V) Developing inflorescences in wild-type plants at the primary branch initiation stage (U) and secondary branch initiation to spikelet initiation
stage (V).
(W) to (Z) Continuous shoot production in MADS14;15;18i pap2-1 quadruple knockdown plants. Shoots and axillary buds are indicated by circles and
arrowheads, respectively. Leaves were removed to show the shoot apices in (U) to (Z).
B, bract; FM, floral meristem; LP, leaf primordia; PB, primary branch; PH, panicle hair; S, spikelet. Bars = 200 mm in (K) to (T), 100 mm in (U) to (W), 1 mm
in (X) and (Y), and 1 cm in (Z).
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MADS14;15;18i pap2-1/+ plants (see Supplemental Figure 7
online). This implies that, among the four genes, PAP2 function
alone is sufficient for normal inflorescence development. However, because upregulation of PAP2 expression was observed in
some of the triple knockdown lines (see Supplemental Figure 5
online), we cannot rule out a possibility that the increased PAP2
activity compensated for the reduction of AP1/FUL expression
in triple knockdown lines.
We observed a slight delay in flowering in the MADS14;15;18i
wild-type and MADS14;15;18i pap2-1/+ plants (Figure 5A). To
quantify the delay, transgenic plants of the T0 generation were
grown under LD conditions in a growth chamber until emergence of
the eighth leaf and then transferred to SD conditions. We counted
the number of leaves produced after transfer to SD conditions in six
and seven independent transgenic plants of MADS14;15;18i wild
type and MADS14;15;18i pap2-1/+, respectively. The number of
leaves in a single plant from each line and the average leaf number
in each genetic background are shown in Figure 5A. Although the
effects varied among independent transgenic lines, a noticeable
increase in leaf number was observed in plants from transgenic
lines containing MADS14;15;18i. These results indicate a possibility
that the three AP1/FUL-like genes of rice are involved in the regulation of flowering time.
PAP2 and AP1/FUL-Like Genes Function Upstream of Hd3a
and RFT1 in Leaves
To reveal the cause of the delay in flowering time, the expression
levels of the rice flowering genes Hd3a and RFT1 were examined
(Figures 5B and 5C). Quadruple knockdown and pap2-1 plants
were grown under natural LD conditions and then transferred to
a growth chamber under SD conditions. Expression of Hd3a and
RFT1 were induced after the transfer to SD conditions in wild-
type control plants. By contrast, extremely low levels of Hd3a
induction were observed in the quadruple knockdown lines. A
slight decrease in Hd3a induction rate was observed in pap2-1.
Expression levels of RFT1 under the SD conditions were also
lowered in pap2-1 and the quadruple lines compared with that
observed in wild-type plants.
Interaction of MADS Domain Proteins
MADS domain proteins dimerize and may form multimeric
complexes to function (reviewed in Gramzow and Theissen,
2010). Therefore, we examined the interactions among PAP2,
MADS14, MADS15, and MADS18 using the yeast two-hybrid
method (Figure 6A). The entire coding regions of the cDNAs
were fused to the activation domain (pGAD) and binding domain
(pGBD) and tested for interactions. As shown in Figure 6A,
a strong interaction was observed between MADS14 and
MADS15. Weak but significant interactions also occurred between PAP2 and PAP2, PAP2 and MADS14, and PAP2 and
MADS15. This indicates that PAP2 is able to form heterodimers
with MADS14 and MADS15, in addition to forming a homodimer.
To pursue further the possibility that PAP2 interacts with
MADS14 and MADS15, we performed transient coexpression
assays of epitope-tagged PAP2 and MADS14 or MADS15 in
Nicotiana benthamiana leaves. The presence of MADS14-FLAG,
MADS15-FLAG, and PAP2-Myc proteins in total protein samples was confirmed by immunoblotting using anti-Myc antibodies (Figures 6B and 6C). After coimmunoprecipitation using
anti-FLAG antibodies, interactions between MADS14 and PAP2
(Figure 6B) and MADS15 and PAP2 (Figure 6C) were detected
by immunoblotting analysis with an anti-Myc antibody. These
biochemical data indicate that PAP2 has the ability to interact
with MADS14 and MADS15 in vivo.
Figure 5. Delay of Flowering Time in Triple Knockdown Plants.
(A) The numbers of leaves produced by individual plants between transfer to SD conditions and heading. Plants were transferred to SD conditions at the
eight-leaf stage, when the eighth leaf was fully expanded. Data are shown for wild-type (wt) and pap2-1/+ and pap2-1 mutant lines transformed with
either the empty vector or the MADS14;15;18i construct. Each bar in the left panel indicates an independent transgenic line. The average number of
leaves in each genotype is shown in the right panel. Bars and asterisks in the right panel represent SD and significant difference from wild-type
determined by the Student’s t test at P < 0.05, respectively.
(B) and (C) Relative expression levels of Hd3a (B) and RFT1 (C) in leaves of pap2-1 and MADS14;15;18i pap2-1 (quad) plants. RNA was isolated from
plants under LD conditions and 3 d after transfer to SD conditions. Bars represent SD for three biological replications.
Inflorescence Meristem Identity of Rice
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7B). In severe cases, spikelets developed directly from callus
(data not shown). By contrast, when the same rolC-Hd3a:GFP
construct was introduced into a pap2-1 background, the plants
exhibited a novel phenotype (Figures 7C and 7D). Precocious
stem elongation was observed, suggesting transition to reproductive development; however, inflorescence development did
not initiate. Instead, vegetative shoots were repeatedly generated resulting in a bushy appearance. Roots initiated from
each tiller shoot, and tillers were connected with very thin stringlike stems (Figure 7C). Inflorescences eventually initiated in the
rolC-Hd3a:GFP pap2-1 lines. Spikelets in these lines contained
increased numbers of glumes (specialized leaves subtending
flowers), indicating retarded specification of SM identity. These
results indicate that PAP2 function is required to initiate the
precocious inflorescence development induced by Hd3a misexpression, supporting our hypothesis that PAP2 and the three
AP1/FUL-like genes act downstream of Hd3a to promote IM
identity.
DISCUSSION
Regulation of IM Identity in Rice
Figure 6. Interaction among Four MADS Box Proteins.
(A) Yeast two-hybrid interaction assays. Full-length coding regions of
PAP2, MADS14, MADS15, and MADS18 were cloned in pGBKT7. PAP2
or MADS15 were also cloned in pGADT7. After cotransformation of
pGBKT7 and pGAD17 vectors, yeast was grown on nonselective (-LT),
weak selective (-LTH), and strong selective (-LTHA) media. The empty
activation domain vector (empty) was included as a negative control.
(B) and (C) Interaction assay in N. benthamiana leaves using MADS14FLAG, PAP2-Myc, and MADS15-Myc (B) and MADS15-FLAG, PAP2-Myc,
and MADS14-Myc (C). Proteins were coexpressed in N. benthamiana
leaves, and the total protein extracts were subjected to immunoprecipitation (IP) using an anti-FLAG antibody. The resultant immune complexes
were analyzed on immunoblots with anti-Myc antibodies.
Genetic Interaction between Hd3a and PAP2
Although it has been suggested that MADS14 and MADS15 act
downstream of Hd3a/RFT1 in the meristem, their functions in
this pathway have not been clearly elucidated (Komiya et al.,
2008, 2009; Taoka et al., 2011). Misexpression of Hd3a causes
extremely early flowering in rice and a number of other plant
species (Izawa et al., 2002; Tamaki et al., 2007). We took advantage of this phenomenon to test the hypothesis that PAP2
functions downstream of Hd3a in the meristem to establish IM
identity. Reproductive transition was remarkably accelerated by
introduction of the rolC-Hd3a:GFP (for green fluorescent protein)
gene (Tamaki et al., 2007) in wild-type plants (Figures 7A and
Our results show that expression of PAP2, a MADS box gene
belonging to a grass-specific subclade of the SEP subfamily,
and three AP1/FUL-like genes (MADS14, MADS15, and
MADS18) is activated in the meristem at the transition to the
reproductive phase. The knockdown of the three AP1/FUL-like
genes did not significantly affect inflorescence development. On
the other hand, the elimination of PAP2 function in the triple
knockdown plants severely impeded transition of the SAM to the
IM. Because IM identity is established normally in the pap2-1
mutant (Kobayashi et al., 2010), the most likely interpretation of
the results with the quadruple knockdown plants is that IM
identity in rice is determined by the combined actions of the
three AP1/FUL-like genes with PAP2. The negative correlation
between total expression levels of three AP1/FUL-like genes
and the severity observed in inflorescence development probably indicates the redundancy in the functions of the three
genes. However, currently it is unknown whether all four genes
contribute equally in specifying IM identity. The individual contributions of each gene to the regulation of IM identity needs to
be elucidated.
It has been proposed that MADS14 and MADS15 function
downstream of Hd3a and RFT1 to transduce the florigen signal
(Komiya et al., 2008, 2009). More recently, transient assays in
cultured cells were used to show that the transcription of
MADS15 is indeed induced by a complex containing Hd3a,
a 14-3-3 protein, and FD (Taoka et al., 2011). Here, we show that
not only MADS14 and MADS15 but also MADS18 and PAP2
exhibit clear induction in the SAM before any visible change in
meristem morphology. We also confirmed that PAP2, MADS14,
and MADS15 interact with one another in vivo. The alleviation of
the precocious flowering phenotype caused by ectopic overexpression of Hd3a in the pap2-1 background also supports the
notion that PAP2 functions downstream of Hd3a in the meristem. On the basis of these results, we conclude that the four
genes analyzed in this study act in the SAM as downstream
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Figure 7. Genetic Interaction between PAP2 and Hd3a.
(A) A wild-type plant regenerated from callus with no inflorescences.
(B) A wild-type plant transformed with prolC-Hd3a:GFP, of the same age
as that shown in (A), with two inflorescences (yellow arrowheads).
(C) A pap2-1 plant transformed with rolC-Hd3a:GFP, of the same age as
those shown in (A) and (B). Stem elongation is observed but no inflorescences have formed. The thin, elongated stems are marked with
white arrowheads.
(D) Frequencies of phenotypes observed in rolC-Hd3a:GFP/wild type
(wt) and rolC-Hd3a:GFP/pap2-1 transgenic plants. Twenty and 16 independent lines for rolC-Hd3a:GFP/wild type and rolC-Hd3a:GFP/pap21, respectively, were analyzed.
(E) An inflorescence on a rolC:Hd3a/wild-type plant. A few normal
spikelets are present.
(F) An inflorescence on a rolC:Hd3a/pap2-1 plant. Each spikelet contains
an increased number of elongated glumes (white arrowheads).
regulators of the florigen signal and give rise to the phase
change of the SAM to the IM (Figure 8).
Meanwhile, the extensive decline of the Hd3a and RFT1 expression levels in leaves of the knockdown plants under SD
conditions suggests the possibility that the AP1/FUL-like genes
of rice and PAP2 function upstream of Hd3a and RFT1 in leaves.
An attractive scenario to explain these results is that AP1/FULlike genes work in distinct ways in leaves and the SAM to promote reproductive transition (Figure 8). First, AP1/FUL-like
genes induce expression of Hd3a and RFT1 in leaves. Once the
florigen is transported from the leaves to the SAM, transcription
of the AP1/FUL-like genes and PAP2 is induced in the SAM
itself, resulting in the transition of the meristem phase to the IM.
This two-step regulation might help to accelerate meristem
phase change by amplifying the florigen signal.
Currently, it is not clear whether the delay in flowering observed in the triple and quadruple knockdown plants is caused
by the loss of AP1/FUL-like activities in leaves, in the meristem,
or both. Similarly, we cannot rule out a possibility that the failure
in establishing IM identity in the quadruple knockdown plants is
a consequence of the reduced activity of Hd3a and RFT1, florigen genes, in leaves. However, we favor an interpretation that
the meristem defects were caused by the reduced function of
the three AP1/FUL-like and PAP2 genes in the meristem based
on the clear induction of these genes in the SAM upon the
transition. Results of the genetic analysis between PAP2 and
Hd3a also support a model in which Hd3a works upstream of
the four MADS box genes in the control of meristem phase
transition. To obtain a conclusive view, roles of the four MADS
box genes in leaves and the meristem should be experimentally
distinguished by more detailed analysis in the near future.
Our findings that AP1/FUL-like genes work upstream of Hd3a
and RFT1 in leaves are consistent with the report that MADS14
and MADS15 work downstream of Early heading date1, a positive regulator of rice flowering, in leaves (Doi et al., 2004).
However, these results are inconsistent with a previous report
describing that the expression of MADS14 and MADS15 in
leaves is regulated by RFT1 (Komiya et al., 2008). A possible
explanation for this discrepancy is that RFT1 and AP1/FUL-like
genes mutually activate each other’s expression. Temperate
grasses, including wheat (Triticum aestivum) and barley (Hordeum
vulgare), require a period of cold temperature to initiate reproductive transition. In these species, VERNALIZATION1
(VRN1), encoding an AP1/FUL-like subfamily MADS domain
protein, is a critical regulator of the vernalization response and
flowering (Danyluk et al., 2003; Trevaskis et al., 2003; Yan et al.,
2003; Shitsukawa et al., 2007). In wheat leaves, VRN1 functions
upstream of FT (Shimada et al., 2009). It has been proposed
that VRN1 triggers flowering through activation of the daylength
response in leaves and acceleration of phase transition in the
meristem, although the function of VRN1 in the meristem has not
been clearly demonstrated (Greenup et al., 2009). This suggests
that the functions of the AP1/FUL-like genes might be conserved in grass species irrespective of any vernalization requirement for flowering.
Contribution of Grass-Specific Genes to the Evolution of
Grass-Specific Forms
Gene duplication is a source of novel genes. Duplicated genes
acquire new functions by changes in their coding regions and/or
their regulatory sequences. The SEP and AP1/FUL subfamilies
of the MADS box genes probably originated and underwent
multiple duplication events before the diversification of the extant angiosperms (Litt and Irish, 2003; Malcomber and Kellogg,
2005; Malcomber et al., 2006; Preston and Kellogg 2006; Zahn
et al., 2005). The evolution of the SEP and AP1/FUL homologs is
conceivably associated with the subsequent dramatic success
of angiosperms. Further duplication events occurred in the ancestors of grasses, resulting in grass-specific SEP and AP1/FUL
Inflorescence Meristem Identity of Rice
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Figure 8. A Model of the Genetic Interaction between MADS Box Genes Regulating the Specification of IM Identity in Rice.
AP1/FUL-like genes and probably PAP2 induce expression of Hd3a and RFT1 in leaves. Once the florigen is transported from the leaves to the SAM,
transcription of the AP1/FUL-like genes and PAP2 is induced in the SAM. This results in the transition of the vegetative meristem phase to the IM.
genes. Among the five SEP genes in rice, PAP2 is unique in
being transcribed in the SAM upon phase transition. This is in
addition to its expression in the SM and glumes, in which abnormalities are observed in pap2-1 mutants. The importance of
the SEP proteins to mediate multimerization of MADS proteins
has been demonstrated. In Arabidopsis, SEP genes influence
floral meristem identity and floral organ development through
formation of multimeric complexes with other MADS domain
proteins that are expressed in a spatially and temporally regulated manner (de Folter et al., 2005; Immink et al., 2009). In
addition to biochemical analysis, the interaction between SEP
and AP1/FUL MADS box genes has also been demonstrated by
genetic analysis in a variety of species (Ditta et al., 2004; Pelaz
et al., 2001). This indicates that SEP and AP1/FUL proteins
potentially interact when they exist in the same cells. Thus, once
PAP2 acquired specialized machinery allowing it to be transcribed in the SAM upon reproductive transition, its interaction
with SAM-expressed AP1/FUL MADS box genes, such as
MADS14 and MADS15, is expected. This enables PAP2 to be
involved in the regulation of IM identity. This is a clear example of gene duplication leading to diversification in the location and timing of expression, resulting in the creation of
a novel function.
(Kobayashi et al., 2010). PAP2 also functions as a suppressor of
the extra elongation of glumes (Gao et al., 2010; Kobayashi
et al., 2010). In general, leaf growth is extensively suppressed
during reproductive growth. Assuming that suppression of
glumes is likely to be essential to secure the SM identity in grass
inflorescence development, an attractive interpretation of PAP2
function is that it ensures transition to the SM in two ways,
namely, induction of SM identity and suppression of glumes.
The new role of PAP2 as a positive regulator of IM identity revealed in this study further expands the importance of PAP2 in
the regulation of rice inflorescence development. In all three
roles, PAP2 positively regulates the progression of the steps in
inflorescence development. Generation of the PAP2 gene in
grasses may be a molecular innovation in inflorescence development. Although the functions of PAP2 orthologs in other
grass species remain to be analyzed, the maize (Zea mays) and
wheat orthologs MADS4 and AGLG, respectively, exhibit expression patterns similar to that of PAP2, suggesting that their
function may be conserved in grass species (Yan et al., 2003;
Danilevskaya et al., 2008).
METHODS
Sample Collection for LM
The Role of PAP2 in Rice Inflorescence Development
The timing of the meristem phase change is crucial for plant
development, and subtle modifications of this timing readily
result in the emergence of novel morphologies (Prusinkiewicz
et al., 2007). During rice inflorescence development, newly
formed meristems acquire BM or SM identity depending upon
the timing and position of their occurrence. We showed previously that PAP2 functions as a positive regulator of SM identity
Rice (Oryza sativa ssp japonica cv Nipponbare) plants grown in a paddy
field in Tsukuba, Japan (;36°N) under field conditions during the 2008
cultivation season were used. Shoot apices were collected at different
growth stages. The samples were fixed in 75% ethanol and 25% acetate
and embedded in paraffin using a microwave processor (Energy Beam
Sciences) according to Takahashi et al. (2010). The paraffin-embedded
samples were cut into 10-µm-thick sections using a microtome (Leica
RM2255). LM was performed using the Veritas Laser Microdissection
System LCC1704 (Arcturus).
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The Plant Cell
Microarray Analysis
Total RNA was extracted from the collected cells with a Pico-Pure RNA
isolation kit (Arcturus) according to the manufacturer’s protocol. The quantity
and quality of RNAs were checked using the Agilent 2100 Bioanalyzer
(Agilent Technologies). One-color spike mix was added to the total RNA prior
to the labeling reaction, and labeling was performed using a Quick Amp
Labeling Kit, One-Color (Agilent Technologies). The cyanine-3 (Cy3)-labeled
cRNA was purified using the RNeasy mini kit (Qiagen), and the quantity was
checked using a NanoDrop ND-1000 UV-VIS spectrophotomer (NanoDrop
Technologies). The Cy3-labeled cRNA sample was fragmented and hybridized with 4 3 44k microarray of rice (Rice Annotation Project-Data Base)
gene expression microarray slides (Agilent; G2519F#15241) at 65°C for 17 h.
The hybridization and washing were performed according to the manufacturer’s instructions. The slides were scanned on an Agilent G2505B DNA
microarray scanner, and background correction of the Cy3 raw signals was
performed using Agilent’s Feature Extraction software (version 9.5.3.1).
We detected 36,100 independent signals corresponding to 27,201
annotated loci and 340 microRNA precursors annotated in the Rice
Annotation Project-Data Base. We selected 19,881 probes that each had
a raw signal intensity above 50 in at least one of the 24 microarray data.
The numbers of microarrays examined are three for the V1, V2, V/R, R3,
and R4 stages, four for the R1 stage, and five for the R2 stage. The
selected raw signal intensities were subjected to quantile normalization
for interarray comparison and then transformed to a log2 scale using the
R program (http://www.r-project.org/) and the limma package (Smyth,
2005). For redundant probes with the same locus ID, we calculated the
average normalized values and thereby obtained the expression profile for
15,193 genes. The median expression values across the seven stages
were subtracted for each gene, and the gene-normalized values were
assigned as relative expression values.
subsequently transferred to SD conditions (12 h light at 28°C, 12 h dark at
24°C). Meristem samples were treated with acetone on ice for 15 min.
After washing with 100 mM NaPO4 buffer, pH7.0, the samples were incubated in GUS staining solution (100 mM NaPO4 buffer, pH 7.0, 10 mM
EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide,
0.1% Triton X-100, and 0.5 mg/mL 5-bromo-4-chrolo-3-indolyl-b-Dglucuronide) at 37°C in the dark.
Scanning Electron Microscopy
Samples were fixed in 2.5% glutaraldehyde overnight at 4°C and dehydrated in a series of ethanol solutions, and then the final ethanol solution was substituted with 3-methylbutyl acetate. The samples were then
dried at critical point, sputter-coated with platinum, and observed under
a scanning electron microscope (S-4000; Hitachi) at an accelerating
voltage of 10 kV.
Real-Time RT-PCR Analysis
Total RNA was extracted using the Plant RNA Isolation Mini Kit (Agilent
Technologies). After DNase I treatment, first-strand cDNA was synthesized by SuperScript III reverse transcriptase (Invitrogen). RT-PCR was
performed using the primer sets shown in Supplemental Table 1 online.
The relative abundance of mRNAs were measured with a Light Cycler 480
system (Roche Applied Science) and normalized against the expression of
ubiquitin. For real-time PCR, three replicates were used for each sample.
In Situ Hybridization Analysis
In situ hybridization of the APO1 and PAP2 genes was performed as
previously described (Ikeda-Kawakatsu et al., 2009; Kobayashi et al., 2010).
Vector Construction
A 520-bp fragment from MADS14 containing significantly high homology
with MADS15 and MADS18 was used to generate the RNAi trigger. This
fragment was amplified using the primers OM14i-110XbaF and OM14i630XbaR (see Supplemental Table 1 online) and then cloned into the
pZH2Bi KXB SpeBam vector (Kuroda et al., 2010) in a tail-to-tail orientation flanking the intron from a rice aspartic protease gene, resulting in
the vector pOsMADS14;15;18i. Similarly, 67-, 77-, and 64-bp fragments
were used as RNAi triggers to generate single knockdown lines of
MADS14, MADS15, and MADS18, respectively. These fragments were
amplified using the primer sets OM14iXba-F/OM14iXba-R (MADS14),
OM15iXba-F/OM15iXba-R (MADS15), and OM18iXba-F/OM18iXba-R
(MADS18) (see Supplemental Table 1 online) and then cloned into pZH2Bi
KXB SpeBam in a tail-to-tail orientation flanking the intron from a rice
aspartic protease gene, resulting in the vectors pMADS14i, pMADS15i,
and pMADS18i, respectively. To construct pPAP2:GUS, a 2.2-kb PAP2
promoter region was amplified using the primers shown in Supplemental
Table 1 online. This fragment was cloned into the pENTR/D-TOPO vector
(Invitrogen). The resulting vector was inserted pGWB3 by the LR recombination reaction (Invitrogen) (Nakagawa et al., 2007).
Rice Transformation
Rice transformation was performed as described by Hiei et al. (1994).
Wild-type (cv Nipponbare) and pap2-1 (Kobayashi et al., 2010) seeds were
used to induce callus for transformation. Hygromycin-selected T1 generation transgenic plants were grown in a growth chamber.
GUS Staining
Transgenic plants containing the PAP2:GUS gene were grown under LD
conditions (14 h light at 28°C, 10 h dark at 24°C) in a chamber for 7 d and
Yeast Two-Hybrid Analysis
The coding regions of PAP2, MADS14, MADS15, and MADS18 were
amplified using the primer sets shown in Supplemental Table 1 online,
cloned into pTA2 (TOYOBO) by TA cloning, and then transferred to the yeast
two-hybrid vector pGBKT7 containing the GAL4 DNA binding domain and
the TRP1 gene. PAP2 and MADS15 were cloned into pGADT7 containing
the GAL4 activation domain and LEU2. Plasmids were cotransformed into
the AH109 strain of Saccharomyces cerevisiae with the Matchmaker twohybrid system (Clontech). Two-hybrid interactions were assayed on selective synthetic defined media, lacking Leu and TRP (-LT), Leu, Trp, and His
(-LTH), or Leu, TRP, His, and adenine (-LTHA).
Transient Expression in Nicotiana benthamiana and
Coimmunoprecipitation Assays
To express the tag-fused proteins, the coding regions of PAP2, MADS14,
and MADS15 were amplified using the primer sets shown in Supplemental
Table 1 online and cloned into pENTR/D-TOPO (Invitrogen), then cloned
into pGWB11 (35S promoter, C-FLAG) or pGWB17 (35S promoter,
C-4xMyc) using LR clonase mix (Invitrogen) (Nakagawa et al., 2007). The
expression constructs were introduced into Agrobacterium tumefaciens
strain EHA105. For transient expression, Agrobacterium strains carrying
each construct were infiltrated into leaves of 3- to 4-week-old N. benthamiana as previously described (Kinoshita et al., 2010). Protein was
extracted from N. benthamiana leaves 3 d after infiltration. Total protein
was extracted according to Gregis et al. (2009). Both total and immunoprecipitated protein were analyzed by SDS-PAGE and immunoblotting using
anti-FLAG (anti-ECS [DDDDK]) (Bethyl; A190-101A) and anti c-Myc (Bethyl;
A190-104A) antibodies. Goat IgG horseradish peroxidase–conjugated
secondary antibody was used (R and D systems; HAF017).
Inflorescence Meristem Identity of Rice
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
databases under the following accession numbers: Os-MADS34,
Q6Q9H6; Os-MADS18, Q0D4T4; Os-MADS20, Q2QQA3; Os-MADS14,
P0C5B1; and Os-MADS15, Q6Q912.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Phylogenetic Analysis of the FUL/AP1,
AGL6-Like, and SEP Subfamilies of MADS Box Proteins in Grasses.
Supplemental Figure 2. In Situ Hybridization Analysis of APO1
Expression in Meristems at Transition (V/R) Stage.
Supplemental Figure 3. The Overall Profile of Gene Expression during
Meristem Phase Transition.
Supplemental Figure 4. Phenotype of MADS14, MADS15, or
MADS18 Single Knockdown Plants.
Supplemental Figure 5. Relative Expression Levels of MADS14,
MADS15, MADS18, MADS20, and PAP2 in Transgenic Plants Containing an OsMADS14;15;18iRNAi Construct.
Supplemental Figure 6. Correlation between the Reduction of
Expression Levels and the Severity of the Phenotype.
Supplemental Figure 7. Inflorescence Development Is Not Affected in
the Triple Knockdown Plants.
Supplemental Table 1. Primers Used in This Study.
Supplemental Data Set 1. Text File of the Alignment Used for the
Phylogenetic Analysis Shown in Supplemental Figure 1.
ACKNOWLEDGMENTS
We thank Ko Shimamoto for prolC-Hd3a:GFP, Yasufumi Daimon for
phylogenetic analysis, and Rebecca Harcourt for critical reading of the
article. This work was supported in part by Grants-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan (22119008,
22247004, and 21027012 to J.K. and 23580012 to H.Y.), by the Ministry of
Agriculture, Forestry and Fisheries (Genomics for Agricultural Innovation; IPG0001 to J.K. and N.Y.), by Genomics for Agricultural Innovation (RTR0002 to
Y.S. and Y.N.), by Genomics for Agricultural Innovation (GAM-207 to H.Y.),
and by the Program for Promotion of Basic Research Activities for Innovation
Bioscience (PROBRAIN; to J.K.).
AUTHOR CONTRIBUTIONS
K.K., N.Y., Y.S., M.Y., R.Y., M.K., H.Y., Y.N., and J.K. designed and
performed the research. K.K., Y.S., and J.K. analyzed data. J.K. wrote
the article.
Received February 15, 2012; revised April 10, 2012; accepted April 21,
2012; published May 8, 2012.
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Inflorescence Meristem Identity in Rice Is Specified by Overlapping Functions of Three AP1/FUL
-Like MADS Box Genes and PAP2, a SEPALLATA MADS Box Gene
Kaoru Kobayashi, Naoko Yasuno, Yutaka Sato, Masahiro Yoda, Ryo Yamazaki, Mayumi Kimizu,
Hitoshi Yoshida, Yoshiaki Nagamura and Junko Kyozuka
Plant Cell; originally published online May 8, 2012;
DOI 10.1105/tpc.112.097105
This information is current as of June 16, 2017
Supplemental Data
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