RESEARCH REPORT 3013
Development 135, 3013-3019 (2008) doi:10.1242/dev.024273
Floral meristem initiation and meristem cell fate are
regulated by the maize AP2 genes ids1 and sid1
George Chuck1,*, Robert Meeley2 and Sarah Hake1
Grass flowers are organized on small branches known as spikelets. In maize, the spikelet meristem is determinate, producing one
floral meristem and then converting into a second floral meristem. The APETALA2 (AP2)-like gene indeterminate spikelet1 (ids1) is
required for the timely conversion of the spikelet meristem into the floral meristem. Ectopic expression of ids1 in the tassel,
resulting from a failure of regulation by the tasselseed4 microRNA, causes feminization and the formation of extra floral meristems.
Here we show that ids1 and the related gene, sister of indeterminate spikelet1 (sid1), play multiple roles in inflorescence
architecture in maize. Both genes are needed for branching of the inflorescence meristem, to initiate floral meristems and to
control spikelet meristem determinacy. We show that reducing the levels of ids1 and sid1 fully suppresses the tasselseed4
phenotype, suggesting that these genes are major targets of this microRNA. Finally, sid1 and ids1 repress AGAMOUS-like MADS-box
transcription factors within the lateral organs of the spikelet, similar to the function of AP2 in Arabidopsis, where it is required for
floral organ fate. Thus, although the targets of the AP2 genes are conserved between maize and Arabidopsis, the genes themselves
have adopted novel meristem functions in monocots.
INTRODUCTION
Meristems provide cells for all the tissues and organs of the plant, and
understanding their activity is essential to decipher the evolution of
plant architecture (Sussex and Kerk, 2001). Meristems can be
classified as determinate or indeterminate based on whether they
terminate in the production of primordia. Inflorescence meristems
(IMs), such as those of racemes, are usually indeterminate, producing
an open number of lateral meristems. Floral meristems (FMs), by
contrast, are usually determinate and terminate in the production of
floral organs. The switch from indeterminacy to determinacy may also
be considered in the light of developmental timing, or heterochrony.
For example, if a switch to determinacy is delayed, the meristem will
initiate extra lateral organs before terminating.
Inflorescence architecture in monocots is dependent upon the
initiation of specific meristem types (McSteen et al., 2000). In
maize, several lateral meristems with defined branching activity are
initiated before FMs are made. These include branch meristems
(BMs), spikelet pair meristems (SPMs) and spikelet meristems
(SMs). Spikelets, the fundamental floral unit of all monocots
(Clifford, 1987), consist of two bract leaves, called glumes, that
enclose a variable number of florets. Maize SPM and SM are
determinate. The SPM produces one SM and then terminates its
activity by differentiating into an SM. Similarly, the SM produces
one FM and then terminates by becoming an FM.
Several maize and rice mutants have provided clues as to how
meristem fates are acquired. SM identity is controlled by the action
of the orthologous genes branched silkless1 (bd1) in maize (Chuck
et al., 2002) and frizzy panicle1 (fzp1) in rice (Komatsu et al., 2003).
In bd1 and fzp1 mutants, the spikelet meristem is replaced by an
indeterminate branch meristem. The bd1 and fzp1 genes encode
1
Plant Gene Expression Center, United States Department of Agriculture –
Agriculture Research Service and the University of California, Albany, CA 94710,
USA. 2Pioneer – A DuPont Company, Johnston, IA 50131, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 9 July 2008
members of the ERF family of APETALA2 (AP2) transcription
factors, and function to repress indeterminate lateral branch
meristem fates. SM determinacy is controlled by another AP2
transcription factor, encoded by the indeterminate spikelet1 (ids1)
gene (Chuck et al., 1998). ids1 mutant spikelets are indeterminate
and initiate extra florets instead of two. In rice, a similar phenotype
is seen in the supernumerary bract (snb) mutant, which delays the
floral transition and produces extra glumes before initiating florets.
snb encodes an AP2 transcription factor similar to ids1, and might
represent a paralogous gene (Lee et al., 2006). ids1 is targeted by the
MIR172 microRNA encoded by the tasselseed4 (ts4) gene (Chuck
et al., 2007). Mutations within the microRNA binding site of ids1
give rise to the dominant Tasselseed6 (Ts6) allele, which affects
spikelet meristem branching and sex determination of floral organs
(Chuck et al., 2007). Double mutant analysis has shown that ids1
mutants suppress the ability of the ts4 SM to initiate ectopic FMs.
Thus, ids1 has diverse functions, affecting sex determination as well
as meristem determinacy.
The orthologous floral homeotic genes LEAFY (LFY) of
Arabidopsis and FLORICAULA (FLO) of Antirrhinum play major
roles in the specification of FM identity in these diverse species
(Weigel et al., 1992; Coen et al., 1990). Flowers on lfy mutants have
characteristics of both floral and inflorescence meristems (Schultz
and Haughn, 1991; Huala and Sussex, 1992; Weigel et al., 1992),
whereas flowers on flo mutants are complete transformations to
inflorescences. LFY activates expression of the MADS-box genes
APETALA3 (Lamb et al., 2002) and AGAMOUS (Hong et al., 2003),
both of which function in specifying floral organ identity. Mutations
in the maize LFY/FLO orthologs, zfl1 and zfl2, affect floral organs
and tassel branch number (Bomblies et al., 2003). Knock-down of
the rice LFY/FLO ortholog, RFL, affects flowering time and panicle
branching, yet spikelets and florets are still made (Rao et al., 2008).
Here we describe a paralog of ids1, sister of indeterminate
spikelet1 (sid1), in maize. sid1 functions together with ids1 to
specify the fate of several lateral meristems in the inflorescence.
Fewer branches form in the tassel and fewer rows of spikelet pairs
form in the ear. Both ear and tassel spikelet meristems are
DEVELOPMENT
KEY WORDS: AP2, Maize, Meristem, miR172
indeterminate and produce supernumerary organs. The tassel
spikelets reiteratively produce bracts and never produce sex organs.
The spikelets in the ear also produce many bracts, but eventually
terminate in an ovule-like structure. Late-forming bracts in the ear
are feminized owing to the ectopic expression of AGAMOUS-like
genes in maize. Thus, ids1 and sid1 are needed to promote
determinacy and produce sex organs, similar to LFY/FLO function
in Arabidopsis and Antirrhinum.
MATERIALS AND METHODS
Phylogenetic analysis
Phylogenetic analysis was performed using MrBayes v. 3.1.2 (Ronquist and
Huelsenbeck, 2003) Markov chain Monte Carlo (MCMC) algorithm using
the Time-Reversible model (GTR) with a proportion of invariable sites (I)
and a gamma distribution parameter (G). The analysis was run for 10,000
generations, sampling trees every 10 generations; 250 trees were discarded
as burn in. Tree shows average branch lengths and posterior probabilities for
all resolved nodes.
Expression analysis
Poly(A)+ RNA gel blots and in situ hybridization were performed as
described (Chuck et al., 2007). For sid1 in situ hybridizations, a 3⬘ fragment
outside the AP2 domain and including the 3⬘ UTR (bp 1301-1730) was used.
This same fragment was used to probe northern blots. Probes for kn1
(Jackson et al., 1994), bd1 (Chuck et al., 2002) and zag1 (Schmidt et al.,
1993) were used as previously described.
Cleavage assay
Poly(A)+ RNA (200 ng) was isolated from 0.5 cm ear primordia from B73
and ts4-TP and used directly for ligation to the GeneRacer 5⬘-RACE oligo
(Invitrogen) and reverse transcribed. One microliter of the reverse
transcription reaction was used for RT-PCR using the GeneRacer 5⬘-RACE
oligo in combination with the SID1 53R oligo (5⬘-AACCATACCCAACAGTGGCGACTG-3⬘) located 280 bases from the microRNA
binding site. The wild-type cleavage product was cloned into pGEM-T Easy
(Promega) and sequenced using M13 primers.
Double mutants
Double mutants between ids1-mum1 and sid1-mum4, between ids1-Burr and
sid1-mum3, and between ids1-mum1, sid1-mum1 and ts4-A were made by
selfing heterozygotes. Homozygotes for sid1 were scored by PCR using sid1
oligos 607 and 608 (5⬘-ATCAGCGTGTGCCTAGCATTTCTTCCCT-3⬘
and 5⬘-CAGTCCCTGCACAATTGGACGACACA-3⬘). ids1 homozygotes
were scored using ids1 oligos 804 and 674 (5⬘-ATCGCAGCTCGATCGTATG-3⬘ and 5⬘-TACAGGGGCGTCACCTTCTA-3⬘). ts4-A
homozygotes were scored using ts4 oligos 7F and 7R (Chuck et al., 2007).
RESULTS AND DISCUSSION
sid1 is targeted by miR172
Previous work identified five potential MIR172 targets (Chuck et al.,
2007). AY109248.1, now referred to as sid1, had high sequence
similarity to ids1, especially within the AP2 domain, where it
displayed 96% amino acid identity (Fig. 1A). A Bayesian
phylogenetic tree of MIR172 targets in rice and maize showed that
sid1 shares a common ancestor with ids1 (Fig. 1B), and is likely to
be orthologous to the rice snb gene, which controls the SM-to-FM
transition (Lee et al., 2006). To determine the function of sid1,
Mutator (Mu) transposons were employed to generate loss-offunction alleles (Bensen et al., 1995). Four Mu insertions were
isolated in sid1, three in exons and one in an intron (Fig. 1C). sid1mum1 and sid1-mum2 alleles had no transcript, whereas the sid1mum3 and sid1-mum4 alleles had longer transcripts, possibly owing
to alternative splicing of the Mu element (Fig. 1F).
A combination of RT-PCR and RNA gel blots was performed
to determine sid1 transcript levels in the wild type and in ts4 and
ids1 mutants. RT-PCR demonstrated that sid1 is widely expressed
Development 135 (18)
in all tissues assayed, including roots, leaves and inflorescences,
and is elevated in tassels from ts4 mutants (Fig. 1D). An increase
in sid1 transcript levels was also detected by gel blots with RNA
from ts4 mutant tassels (Fig. 1F) and ears (Fig. 1G), indicating
that the sid1 transcript might be subject to negative regulation by
ts4. In support of this, RNA cleavage assays of sid1 transcripts
from ts4-TP mutants, as compared with those from wild type,
showed differences in the amount of cleavage within the
microRNA binding site. In ts4-TP mutants, most transcripts were
cleaved 3⬘ of the microRNA binding site (Fig. 1E). By contrast,
in wild-type RNA, almost all transcripts were cleaved between bp
10 and 11 of the microRNA binding site, as expected. Some
cleavage of sid1 still occurred in ts4-TP within the microRNA
binding site, suggesting that sid1 is also targeted by other
members of the MIR172 family, of which there are at least four
(Chuck et al., 2007). sid1 expression was also increased in ids1
mutant ears as compared with wild type (Fig. 1G). This finding
indicates that sid1 might be under negative regulation by ids1.
AP2 genes are predicted to be negatively autoregulated in both
Arabidopsis (Schwab et al., 2005) and wheat (Simons et al.,
2006). Compensation between redundant genes has been
documented in numerous cases, for example with the PIN genes
in Arabidopsis. Mutations in pin7 result in ectopic expression of
PIN4, and PIN1 is ectopically induced in pin2 mutants (Vieten et
al., 2005).
The function of ids1 and sid1 in spikelet meristem
fate
Maize is a monoecious plant, in which male and female flowers are
borne on distinct inflorescences. Sex determination occurs through
abortion of carpel primordia in the tassel and arrest of stamen
primordia in the ear (Cheng et al., 1983). Ears and tassels also differ
by the presence of BMs, which form side branches in the tassel and
are absent from the ear (Fig. 2A, Fig. 3A). SPMs initiate in ordered
rows in a spiral phyllotaxy from the inflorescence meristem and in
a distichous phyllotaxy along the tassel branches (Fig. 3A,B). Each
SPM initiates an SM and converts to a second SM. Each SM initiates
two sterile leaves called glumes, followed by two lemmas, each
containing FMs in their axils. The FM initiates a palea, lodicules,
stamens and finally a pistil, or silk (Fig. 3C,D). In tassels, the pistils
abort to produce a pair of staminate florets (Fig. 2H), whereas in the
ear, the lower floret and the stamens abort to produce a single
pistillate floret (Fig. 2D) (Cheng et al., 1983).
Since sid1 mutants appeared to be phenotypically normal (data
not shown), double mutants with ids1 were analyzed. These double
mutants affected all the lateral meristems of the inflorescence. The
tassels of the ids1-Burr;sid1-mum3 double mutant (Fig. 2A) had
fewer branches than with ids1-Burr alone (Kaplinsky and Freeling,
2003). Normal female inflorescences initiate seven to eight rows of
SPMs, but ids1-Burr;sid1-mum3 double mutants initiated a
maximum of four rows, and often had bare rachis in place of the
missing rows (Fig. 2B). Although the ears of the double mutant
appeared to initiate silks, seed set was reduced 5- to 8-fold compared
with ids1-Burr (Fig. 2C), indicating that floral organ function was
affected. In fact, most of the seeds that formed failed to germinate.
ids1-Burr ear spikelets initiate extra lateral florets in the axils of
lemmas (Fig. 2E). In the double mutant, these extra lateral florets
were absent, and the spikelet terminated in a floret-like structure
(Fig. 2F,G). This terminal structure was enclosed by several bracts
that have silk-like characteristics at their tips (Fig. 2G). ids1-Burr
tassel spikelets also initiate several functional florets (Fig. 2I). The
tassels of the double mutant, however, did not initiate any florets,
DEVELOPMENT
3014 RESEARCH REPORT
ids1 and sid1 regulate meristem fates
RESEARCH REPORT 3015
and continuously initiated bracts instead (Fig. 2J). A double mutant
between different alleles, ids1-mum1 and sid1-mum4, appeared
identical to ids1-Burr;sid1-mum3 (data not shown), and thus these
phenotypes are not allele-specific.
Although ids1 is targeted by ts4, ids1 mutants do not completely
suppress the ts4 mutant phenotype because the ear of the double
mutant still resembles that of ts4 single mutants (Fig. 2K) (Chuck et
al., 2007). Thus, at least one other target gene must be responsible
for the continued presence of the ts4 ear phenotype in the ids1;ts4
double mutant. Double mutants between sid1-mum1 and ts4-A
resembled ts4-A alone in both the tassel (Fig. 2L) and ear (Fig. 2N,
left). However, when these double mutants were also made
heterozygous for ids1-mum1, the tassel was normalized and
resembled a weak ts4 mutant phenotype (Fig. 2M), whereas the ear
resembled that of wild type (Fig. 2N, right). This result supports the
RNA analysis (Fig. 1D,F,G) and indicates that sid1 is a ts4 target
gene, although its suppressive effects can only be observed in
combination with ids1. The ids1-mum1;sid1-mum1;ts4-A triple
mutant resembled the ids1-Burr;sid1-mum3 double mutant and
completely suppressed the ts4 sex-determination phenotype in the
tassel (Fig. 2A, right) and extra branching phenotypes in the ear
(data not shown). Thus, ids1 and sid1 together are epistatic to ts4.
Scanning electron microscopy was used to examine the
ids1;sid1 double mutant phenotype more closely. ids1-mum1 ear
tips normally initiate six rows of spikelet pair meristems (Fig.
3E). ids1-mum1 ear spikelets normally initiate several extra
lemmas, each of which contains FMs in their axils (Fig. 3F).
These extra FMs are fully functional, and initiate floral organs in
a normal pattern (Fig. 3G). By contrast, ids1-Burr;sid1-mum3
tassels initiated fewer BMs (Fig. 3H), and only four rows of SPM
DEVELOPMENT
Fig. 1. Molecular analysis of maize sid1. (A) Amino acid alignment of maize SID1 (top) and IDS1 (bottom). Black line marks the AP2 domain.
(B) Bayesian phylogram of DNA sequences of the AP2 domains of all MIR172-targeted genes from maize and rice. The Arabidopsis AP2 gene was
used as the outgroup. (C) Genomic structure of the maize sid1 gene. Boxes represent exons, dark boxes represent the AP2 domain, and triangles
represent Mutator transposon insertions. (D) RT-PCR of sid1 (top) and actin (bottom) from different tissues and tassels of ts4 mutant alleles.
(E) Location of cleavage sites within sid1 from B73 ears (top) and ts4-TP ears (bottom). The number of clones cleaved at each different position
within the microRNA binding site over the total number of clones analyzed is indicated; those in parentheses to the right indicate the number of
clones cleaved 3⬘ of the microRNA binding site. (F) RNA gel blot using 1 μg of poly(A)+ RNA from 0.5 cm tassels. Arrowheads mark positions of the
28S and 18S ribosomal RNAs. (G) RNA gel blot using 1 μg of poly(A)+ RNA from 0.5 cm ears. The 3⬘ ends of ids1 and sid1 outside of the coding
region for the AP2 domain were used as probes.
3016 RESEARCH REPORT
Development 135 (18)
Fig. 2. sid1/ids1 mutant phenotypes. (A) Tassels of maize ids1-Burr
(left), sid1-mum3;ids1-Burr (middle) and ids1-mum1;sid1-mum1;ts4-A
triple (right) mutants. (B) Mature ear of sid1-mum3;ids1-Burr. Bare
rachis is present in place of missing rows. (C) Fertilized ears of sid1mum3;ids1-Burr (left) compared with ids1-Burr (right). (D) Hand section
of wild-type (WT) ear spikelet. (E) Hand section of ids1-mum1 ear
spikelet. Extra florets are arranged laterally in the axils of bracts.
(F) Hand section of sid1-mum3;ids1-Burr ear spikelet. Floret-like
structure is terminal. (G) Isolated bract surrounding ovule of sid1mum3;ids1-Burr ear spikelet showing silk transformation at tip.
(H) Wild-type tassel spikelet showing two florets (arrowheads). (I) ids1mum1 tassel spikelet with three florets (arrowheads). (J) sid1mum3;ids1-Burr tassel spikelet showing absence of florets. (K) ts4A;ids1-Burr double mutant tassel (left) and ear (right). The tip of the ear
is highly branched. (L) Tassel of ts4-A;sid1-mum1 double mutant.
(M) Tassel of plant that is homozygous for ts4-A and sid1-mum1 but
heterozygous for ids1-mum1. (N) Ear of ts4-A;sid1-mum1 double
mutant (left) and of ts4-A;sid1-mum1 double mutant heterozygous for
ids1-mum1 (right). Plants are siblings.
(Fig. 3I). The SM of the tassel in the double mutant was
indeterminate and continuously initiated lemma-like bracts with
no FMs (Fig. 3J). The ear of the double mutant also initiated fewer
rows (Fig. 3K, Fig. 4G). In contrast to the tassel, the SM of the ear
terminated in an ovule-like structure after initiating several bracts
(Fig. 3L). No stamens or lodicules were observed, indicating that
the normal pattern of floral organ initiation is not followed in the
double mutant. Later in development, the tips of the bracts began
to differentiate structures that resemble silks (Fig. 3M).
Expression patterns of meristem markers suggest
that the SM never transitions to FM fate in
ids1;sid1 mutants
In situ hybridization using meristem-specific markers was carried
out to further understand the basis for the indeterminacy and organ
identity defects in ids1;sid1 double mutants. The maize knotted1
(kn1) gene is expressed in the indeterminate cells of the meristem
and is downregulated upon organ initiation (Jackson et al., 1994).
Cross-sections of ids1-Burr ears compared with ids1-Burr;sid1mum3 ears showed that the number of rows of SPMs was reduced
(Fig. 4, compare F with G). kn1 expression in the spikelets of the
double mutant persisted after the initiation of several sterile bracts
(Fig. 4H), indicating that the meristem is indeterminate. The
branched silkless1 (bd1) gene is expressed in a semi-circular domain
at the base of the SM and disappears upon floret initiation in maize
and rice (Chuck et al., 2002). In young ids1-Burr;sid1-mum3
spikelets, the bd1 expression pattern appeared normal (Fig. 4I).
Later in development, however, instead of disappearing, this
expression pattern persisted (Fig. 4J), demonstrating that the
meristem had retained SM identity longer than normal.
The maize AGAMOUS-like MADS-box genes zmm2 and zag1 are
duplicate genes that mark the FM as well as stamens and carpels
(Schmidt et al., 1993; Mena et al., 1996) (Fig. 4K,M). They are not
expressed in leaf-like organs, such as palea or lemma. zag1
mutations cause extra carpels to form that fail to fuse into a
functional silk (Mena et al., 1996). At early stages of spikelet
development, we saw no expression of zmm2 (data not shown) or
zag1 (Fig. 4N, inset) in the ids1;sid1 double mutant. However,
ectopic zmm2 expression was observed later in the SM of the double
mutant as well as in the carpelloid bracts near the apex (Fig. 4L).
zag1 was ectopically expressed in these same regions (Fig. 4N). The
ectopic expression of AGAMOUS-like MADS-box genes in the
ids1;sid1 double mutant is consistent with the role of the
Arabidopsis AP2 gene as a negative regulator of AGAMOUS (Drews
et al., 1991). Finally, the maize AP3 ortholog, silky1, which is
necessary for stamen and lodicule patterning (Ambrose et al., 2000),
was not expressed in the double mutant at early (not shown) or late
(Fig. 4P) stages, whereas it was detected in the stamens or lodicules
of wild-type controls (Fig. 4O).
Based on previously described differences between the ts4 and
Ts6 mutant phenotypes (Chuck et al., 2007), it was postulated that at
least one other gene was targeted by the ts4 microRNA in addition
to ids1. sid1 appears to be that gene based on four criteria: its
expression levels are elevated in ts4 mutants (Fig. 1D,F,G); its
cleavage is reduced in a ts4 mutant background (Fig. 1E); it is
ectopically expressed in ts4 mutant inflorescences (Fig. 4E); and
sid1 mutants enhance suppression of the ts4 mutant phenotype by
ids1-mum1 (Fig. 2N). Previous work showed that MIR172 represses
DEVELOPMENT
Expression of sid1 in the wild type and ids1 and
ts4 mutants
To examine the expression of sid1 more precisely, in situ
hybridization on wild-type and mutant tissue was performed. sid1
transcript was present in both the SPM and SM of wild type (Fig.
4A), but was absent from the sid1-mum1 inflorescence (Fig. 4B).
After florets were initiated, sid1 transcript was found in all floral
organs, including the carpels and stamens, as well as the lower FM
(Fig. 4C). In ids1-mum1 florets, a similar pattern was seen (Fig. 4D),
although expression was expanded owing to the initiation of extra
meristems. The ts4 ear tip showed ectopic sid1 expression near the
inflorescence meristem tip, as well as higher-level expression in the
lateral meristems (Fig. 4E).
ids1 and sid1 regulate meristem fates
RESEARCH REPORT 3017
its AP2 target genes at the level of translation (Chen, 2004;
Aukerman and Sakai, 2003). Other groups have found that MIR172
repression may act at both the level of translation and transcript
cleavage (Schwab et al., 2005). We showed that IDS1 is regulated
at the level of translation by ts4 (Chuck et al., 2007). Our analysis of
sid1 suggests that it is regulated at the level of transcript stability,
although we cannot rule out an additional level of translational
regulation. Thus, it is likely that ts4 regulates AP2 transcripts using
both modes of regulation.
Lack of negative regulation of ids1 in Ts6 mutants leads to SMs
that initiate extra florets in a random phyllotaxy (Irish, 1997; Chuck
et al., 2007). Surprisingly, ids1 mutants also initiate extra florets, but
in a defined distichous phyllotaxy. Thus, the loss-of-function and
gain-of-function phenotypes for ids1 seem similar with regard to
floret initiation. This conflicting result can be explained by the
function of its redundant paralog, sid1, which masks the ids1 lossof-function null phenotype. Loss of both sid1 and ids1 leads to loss
of FM initiation in the tassel and ear. Therefore, the gain-of-function
phenotype of ids1 (Ts6) shows that it is sufficient for FM initiation,
and the complete loss of function of ids1 and sid1 shows that it is
necessary for FM initiation.
Loss of ids1 and sid1 also results in a reduction in the number of
lateral meristems that initiate from the inflorescence meristem. This
result seems to indicate a role for ids1 and sid1 in maintaining the
stem cell niche, much like AP2 genes in Arabidopsis (Aida et al.,
2004; Wurschum et al., 2006). The use of microRNA-resistant forms
of AP2 demonstrated that this property acts through the WUSCHEL
(WUS) pathway, which plays a key role in stem cell maintenance
(Zhao et al., 2007). However, a simple comparison of the
inflorescence meristems of the wild type and ids1;sid1 mutant (Fig.
4A,H) showed that they were approximately the same size. This
indicates that the more likely function of ids1 and sid1 is to regulate
initiation of lateral meristems in the inflorescence, rather than
regulating stem cell maintenance.
The carpelloid bract defects in ids1-Burr;sid1-mum3 ear spikelets
appear to be caused by ectopic expression of AGAMOUS-like
MADS-box genes in the lateral organs of the spikelet. AP2 mutants
in Arabidopsis display carpelloid sepals owing to ectopic expression
of AGAMOUS in those organs (Drews et al., 1991). The ids1;sid1
floral organ phenotype demonstrates that this property is conserved
in maize. Interestingly, the ectopic in situ expression of zag1 and
zmm2 was not observed in the tassel (data not shown), which might
indicate that these genes have divergent functions in the male and
female inflorescences, as previously suggested (Mena et al., 1996).
The ids1;sid1 tassel does not initiate FMs or floral organs. This
function is unique to maize, because ap2 mutants in Arabidopsis
continue to make flowers. A role for ap2 in FM fate is seen in double
mutants with ap1. Arabidopsis ap2;ap1 mutant flowers are less
DEVELOPMENT
Fig. 3. Scanning electron microscopy of wild
type, ids1-mum1 and ids1-mum1;sid1mum3. (A) Wild-type maize tassel, side view.
(B) Wild-type tassel, top view, showing seven
rows of SPM (arrowheads). (C) Wild-type ear
floret with initiating floral organs. Carpels are
growing over the ovule primordium. (D) Older
ear floret showing growing silk composed of
fused carpels. (E) Top view of ids1-Burr ear
showing six SPM rows (arrowheads). (F) ids1-Burr
spikelets with florets in the axils of extra lemma
bracts. (G) Older ids1-Burr spikelet with initiating
floral organs. Floral organs initiate in a pattern
similar to wild type. (H-M) ids1-Burr;sid1-mum4
double mutant. (H) Tassel primordium with a
single BM (arrowhead). (I) Top view of tassel
showing four rows of SPM (arrowheads). (J)
Indeterminate male spikelets with multiple bracts
and no florets. (K) Young ear primordium with
four rows of SPM (arrowheads). (L) Female
spikelet terminating in an ovule primordium.
(M) Older female spikelet with bract-to-silk
transformations. SPM, spikelet pair meristem;
SM, spikelet meristem; BM, branch meristem; S,
stamen; OP, ovule primordium; Le, lemma bract;
LF, lower floret; Si, silk; F, floret; C, carpel. Scale
bars: 100 μm.
3018 RESEARCH REPORT
Development 135 (18)
Fig. 4. In situ hybridization analysis of wild type, ts4
and ids1;sid1 mutants. Probes are listed at lower left of
each panel, genotypes at lower right. (A-E) sid1 in situs.
(A) Wild-type maize tassel primordium. (B) sid1-mum2
tassel primordium. (C) Wild-type ear florets with initiating
floral organs. (D) ids-mum1 ear florets. (E) ts4-A ear
inflorescence. (F-H) kn1 in situs. (F) Cross-section of ear of
ids1-Burr. Arrowheads indicate rows. (G) Cross-section of
ear of ids1-Burr;sid1-mum3. (H) Close-up of spikelet of
double mutant showing prolonged kn1 expression. (I,J) bd1
in situs. Expression marked with arrowheads.
(I) Longitudinal section of ids1-Burr;sid1-mum3 ear showing
normal expression. (J) Older spikelets of double mutant
showing prolonged expression. (K,L) zmm2 in situs.
(K) Wild-type ear florets with initiating floral organs.
(L) ids1-Burr;sid1-mum3 ear spikelets with ectopic
expression in bracts and meristem. (M,N) zag1 in situs.
(M) Wild-type ear florets with initiating floral organs. (N)
ids1-Burr;sid1-mum3 ear spikelets with ectopic expression
in bracts and meristem. Inset shows a younger stage.
(O,P) silky1 in situs. (O) Wild-type ear florets with initiating
floral organs. (P) ids1-Burr;sid1-mum3 ear spikelets showing
no expression. Lo, lodicules.
transition to FM initiation, then a block at this step could lead to the
formation of multiple sterile organs. Since floral bracts are present
in both Arabidopsis and maize, these pathways are likely to be
parallel in both monocots and dicots. One intriguing possibility is
that the divergence of monocots and dicots has led the AP2 genes to
substitute for LFY function in the grasses. Analysis of sid1 and ids1
in other genera closer to the monocot/dicot divide will elucidate the
evolution of AP2 versus LFY genes in FM fate.
We thank D. Hantz for greenhouse maintenance; B. Thompson, N. Bolduc, C.
Lunde and H. Candela for helpful comments on the manuscript; and Devin
O’Conner for phylogenetic analysis. This work was supported by National
Science Foundation (NSF) grant DBI-0604923 and by USDA-ARS Current
Research Information System (CRIS) grant 5335-21000-018-00D to S.H., and
by Cooperative State Research, Education and Extension Service (CSREES)
grant 2004-35301-14507 to G.C.
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ids1 and sid1 regulate meristem fates