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INTRODUCTIONMeristems provide cells for all the tissues and organs of the plant, andunderstanding their activity is essential to decipher the evolution ofplant architecture (Sussex and Kerk, 2001). Meristems can beclassified as determinate or indeterminate based on whether theyterminate in the production of primordia. Inflorescence meristems(IMs), such as those of racemes, are usually indeterminate, producingan open number of lateral meristems. Floral meristems (FMs), bycontrast, are usually determinate and terminate in the production offloral organs. The switch from indeterminacy to determinacy may alsobe considered in the light of developmental timing, or heterochrony.For example, if a switch to determinacy is delayed, the meristem willinitiate extra lateral organs before terminating.

Inflorescence architecture in monocots is dependent upon theinitiation of specific meristem types (McSteen et al., 2000). Inmaize, several lateral meristems with defined branching activity areinitiated 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, thatenclose a variable number of florets. Maize SPM and SM aredeterminate. The SPM produces one SM and then terminates itsactivity by differentiating into an SM. Similarly, the SM producesone FM and then terminates by becoming an FM.

Several maize and rice mutants have provided clues as to howmeristem fates are acquired. SM identity is controlled by the actionof the orthologous genes branched silkless1 (bd1) in maize (Chucket al., 2002) and frizzy panicle1 (fzp1) in rice (Komatsu et al., 2003).In bd1 and fzp1 mutants, the spikelet meristem is replaced by anindeterminate branch meristem. The bd1 and fzp1 genes encode

members of the ERF family of APETALA2 (AP2) transcriptionfactors, and function to repress indeterminate lateral branchmeristem fates. SM determinacy is controlled by another AP2transcription factor, encoded by the indeterminate spikelet1 (ids1)gene (Chuck et al., 1998). ids1 mutant spikelets are indeterminateand initiate extra florets instead of two. In rice, a similar phenotypeis seen in the supernumerary bract (snb) mutant, which delays thefloral transition and produces extra glumes before initiating florets.snb encodes an AP2 transcription factor similar to ids1, and mightrepresent a paralogous gene (Lee et al., 2006). ids1 is targeted by theMIR172 microRNA encoded by the tasselseed4 (ts4) gene (Chucket al., 2007). Mutations within the microRNA binding site of ids1give rise to the dominant Tasselseed6 (Ts6) allele, which affectsspikelet meristem branching and sex determination of floral organs(Chuck et al., 2007). Double mutant analysis has shown that ids1mutants suppress the ability of the ts4 SM to initiate ectopic FMs.Thus, ids1 has diverse functions, affecting sex determination as wellas meristem determinacy.

The orthologous floral homeotic genes LEAFY (LFY) ofArabidopsis and FLORICAULA (FLO) of Antirrhinum play majorroles in the specification of FM identity in these diverse species(Weigel et al., 1992; Coen et al., 1990). Flowers on lfy mutants havecharacteristics of both floral and inflorescence meristems (Schultzand Haughn, 1991; Huala and Sussex, 1992; Weigel et al., 1992),whereas flowers on flo mutants are complete transformations toinflorescences. LFY activates expression of the MADS-box genesAPETALA3 (Lamb et al., 2002) and AGAMOUS (Hong et al., 2003),both of which function in specifying floral organ identity. Mutationsin the maize LFY/FLO orthologs, zfl1 and zfl2, affect floral organsand tassel branch number (Bomblies et al., 2003). Knock-down ofthe rice LFY/FLO ortholog, RFL, affects flowering time and paniclebranching, yet spikelets and florets are still made (Rao et al., 2008).

Here we describe a paralog of ids1, sister of indeterminatespikelet1 (sid1), in maize. sid1 functions together with ids1 tospecify the fate of several lateral meristems in the inflorescence.Fewer branches form in the tassel and fewer rows of spikelet pairsform in the ear. Both ear and tassel spikelet meristems are

Floral meristem initiation and meristem cell fate areregulated by the maize AP2 genes ids1 and sid1George Chuck1,*, Robert Meeley2 and Sarah Hake1

Grass flowers are organized on small branches known as spikelets. In maize, the spikelet meristem is determinate, producing onefloral meristem and then converting into a second floral meristem. The APETALA2 (AP2)-like gene indeterminate spikelet1 (ids1) isrequired 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 inflorescencearchitecture in maize. Both genes are needed for branching of the inflorescence meristem, to initiate floral meristems and tocontrol spikelet meristem determinacy. We show that reducing the levels of ids1 and sid1 fully suppresses the tasselseed4phenotype, suggesting that these genes are major targets of this microRNA. Finally, sid1 and ids1 repress AGAMOUS-like MADS-boxtranscription factors within the lateral organs of the spikelet, similar to the function of AP2 in Arabidopsis, where it is required forfloral organ fate. Thus, although the targets of the AP2 genes are conserved between maize and Arabidopsis, the genes themselveshave adopted novel meristem functions in monocots.

KEY WORDS: AP2, Maize, Meristem, miR172

Development 135, 3013-3019 (2008) doi:10.1242/dev.024273

1Plant 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: gchuck@nature.berkeley.edu)

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indeterminate and produce supernumerary organs. The tasselspikelets reiteratively produce bracts and never produce sex organs.The spikelets in the ear also produce many bracts, but eventuallyterminate in an ovule-like structure. Late-forming bracts in the earare feminized owing to the ectopic expression of AGAMOUS-likegenes in maize. Thus, ids1 and sid1 are needed to promotedeterminacy and produce sex organs, similar to LFY/FLO functionin Arabidopsis and Antirrhinum.

MATERIALS AND METHODSPhylogenetic analysisPhylogenetic analysis was performed using MrBayes v. 3.1.2 (Ronquist andHuelsenbeck, 2003) Markov chain Monte Carlo (MCMC) algorithm usingthe Time-Reversible model (GTR) with a proportion of invariable sites (I)and a gamma distribution parameter (G). The analysis was run for 10,000generations, sampling trees every 10 generations; 250 trees were discardedas burn in. Tree shows average branch lengths and posterior probabilities forall resolved nodes.

Expression analysisPoly(A)+ RNA gel blots and in situ hybridization were performed asdescribed (Chuck et al., 2007). For sid1 in situ hybridizations, a 3� fragmentoutside 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 assayPoly(A)+ RNA (200 ng) was isolated from 0.5 cm ear primordia from B73and ts4-TP and used directly for ligation to the GeneRacer 5�-RACE oligo(Invitrogen) and reverse transcribed. One microliter of the reversetranscription reaction was used for RT-PCR using the GeneRacer 5�-RACEoligo in combination with the SID1 53R oligo (5�-AACCAT -ACCCAACAGTGGCGACTG-3�) located 280 bases from the microRNAbinding site. The wild-type cleavage product was cloned into pGEM-T Easy(Promega) and sequenced using M13 primers.

Double mutantsDouble mutants between ids1-mum1 and sid1-mum4, between ids1-Burr andsid1-mum3, and between ids1-mum1, sid1-mum1 and ts4-A were made byselfing heterozygotes. Homozygotes for sid1 were scored by PCR using sid1oligos 607 and 608 (5�-ATCAGCGTGTGCCTAGCATTTCTTC CCT-3�and 5�-CAGTCCCTGCACAATTGGACGACACA-3�). ids1 homozygoteswere scored using ids1 oligos 804 and 674 (5�-ATCGCAGC -TCGATCGTATG-3� and 5�-TACAGGGGCGTCAC CT TC TA-3�). ts4-Ahomozygotes were scored using ts4 oligos 7F and 7R (Chuck et al., 2007).

RESULTS AND DISCUSSIONsid1 is targeted by miR172Previous work identified five potential MIR172 targets (Chuck et al.,2007). AY109248.1, now referred to as sid1, had high sequencesimilarity to ids1, especially within the AP2 domain, where itdisplayed 96% amino acid identity (Fig. 1A). A Bayesianphylogenetic tree of MIR172 targets in rice and maize showed thatsid1 shares a common ancestor with ids1 (Fig. 1B), and is likely tobe orthologous to the rice snb gene, which controls the SM-to-FMtransition (Lee et al., 2006). To determine the function of sid1,Mutator (Mu) transposons were employed to generate loss-of-function alleles (Bensen et al., 1995). Four Mu insertions wereisolated in sid1, three in exons and one in an intron (Fig. 1C). sid1-mum1 and sid1-mum2 alleles had no transcript, whereas the sid1-mum3 and sid1-mum4 alleles had longer transcripts, possibly owingto alternative splicing of the Mu element (Fig. 1F).

A combination of RT-PCR and RNA gel blots was performedto determine sid1 transcript levels in the wild type and in ts4 andids1 mutants. RT-PCR demonstrated that sid1 is widely expressed

in all tissues assayed, including roots, leaves and inflorescences,and is elevated in tassels from ts4 mutants (Fig. 1D). An increasein sid1 transcript levels was also detected by gel blots with RNAfrom ts4 mutant tassels (Fig. 1F) and ears (Fig. 1G), indicatingthat the sid1 transcript might be subject to negative regulation byts4. In support of this, RNA cleavage assays of sid1 transcriptsfrom ts4-TP mutants, as compared with those from wild type,showed differences in the amount of cleavage within themicroRNA binding site. In ts4-TP mutants, most transcripts werecleaved 3� of the microRNA binding site (Fig. 1E). By contrast,in wild-type RNA, almost all transcripts were cleaved between bp10 and 11 of the microRNA binding site, as expected. Somecleavage of sid1 still occurred in ts4-TP within the microRNAbinding site, suggesting that sid1 is also targeted by othermembers of the MIR172 family, of which there are at least four(Chuck et al., 2007). sid1 expression was also increased in ids1mutant ears as compared with wild type (Fig. 1G). This findingindicates that sid1 might be under negative regulation by ids1.AP2 genes are predicted to be negatively autoregulated in bothArabidopsis (Schwab et al., 2005) and wheat (Simons et al.,2006). Compensation between redundant genes has beendocumented in numerous cases, for example with the PIN genesin Arabidopsis. Mutations in pin7 result in ectopic expression ofPIN4, and PIN1 is ectopically induced in pin2 mutants (Vieten etal., 2005).

The function of ids1 and sid1 in spikelet meristemfateMaize is a monoecious plant, in which male and female flowers areborne on distinct inflorescences. Sex determination occurs throughabortion of carpel primordia in the tassel and arrest of stamenprimordia in the ear (Cheng et al., 1983). Ears and tassels also differby the presence of BMs, which form side branches in the tassel andare absent from the ear (Fig. 2A, Fig. 3A). SPMs initiate in orderedrows in a spiral phyllotaxy from the inflorescence meristem and ina distichous phyllotaxy along the tassel branches (Fig. 3A,B). EachSPM initiates an SM and converts to a second SM. Each SM initiatestwo sterile leaves called glumes, followed by two lemmas, eachcontaining FMs in their axils. The FM initiates a palea, lodicules,stamens and finally a pistil, or silk (Fig. 3C,D). In tassels, the pistilsabort to produce a pair of staminate florets (Fig. 2H), whereas in theear, the lower floret and the stamens abort to produce a singlepistillate floret (Fig. 2D) (Cheng et al., 1983).

Since sid1 mutants appeared to be phenotypically normal (datanot shown), double mutants with ids1 were analyzed. These doublemutants affected all the lateral meristems of the inflorescence. Thetassels of the ids1-Burr;sid1-mum3 double mutant (Fig. 2A) hadfewer branches than with ids1-Burr alone (Kaplinsky and Freeling,2003). Normal female inflorescences initiate seven to eight rows ofSPMs, but ids1-Burr;sid1-mum3 double mutants initiated amaximum of four rows, and often had bare rachis in place of themissing rows (Fig. 2B). Although the ears of the double mutantappeared to initiate silks, seed set was reduced 5- to 8-fold comparedwith ids1-Burr (Fig. 2C), indicating that floral organ function wasaffected. In fact, most of the seeds that formed failed to germinate.ids1-Burr ear spikelets initiate extra lateral florets in the axils oflemmas (Fig. 2E). In the double mutant, these extra lateral floretswere absent, and the spikelet terminated in a floret-like structure(Fig. 2F,G). This terminal structure was enclosed by several bractsthat have silk-like characteristics at their tips (Fig. 2G). ids1-Burrtassel spikelets also initiate several functional florets (Fig. 2I). Thetassels of the double mutant, however, did not initiate any florets,

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and continuously initiated bracts instead (Fig. 2J). A double mutantbetween different alleles, ids1-mum1 and sid1-mum4, appearedidentical to ids1-Burr;sid1-mum3 (data not shown), and thus thesephenotypes are not allele-specific.

Although ids1 is targeted by ts4, ids1 mutants do not completelysuppress the ts4 mutant phenotype because the ear of the doublemutant still resembles that of ts4 single mutants (Fig. 2K) (Chuck etal., 2007). Thus, at least one other target gene must be responsiblefor the continued presence of the ts4 ear phenotype in the ids1;ts4double mutant. Double mutants between sid1-mum1 and ts4-Aresembled ts4-A alone in both the tassel (Fig. 2L) and ear (Fig. 2N,left). However, when these double mutants were also madeheterozygous for ids1-mum1, the tassel was normalized andresembled a weak ts4 mutant phenotype (Fig. 2M), whereas the earresembled that of wild type (Fig. 2N, right). This result supports the

RNA analysis (Fig. 1D,F,G) and indicates that sid1 is a ts4 targetgene, although its suppressive effects can only be observed incombination with ids1. The ids1-mum1;sid1-mum1;ts4-A triplemutant resembled the ids1-Burr;sid1-mum3 double mutant andcompletely suppressed the ts4 sex-determination phenotype in thetassel (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 theids1;sid1 double mutant phenotype more closely. ids1-mum1 eartips normally initiate six rows of spikelet pair meristems (Fig.3E). ids1-mum1 ear spikelets normally initiate several extralemmas, each of which contains FMs in their axils (Fig. 3F).These extra FMs are fully functional, and initiate floral organs ina normal pattern (Fig. 3G). By contrast, ids1-Burr;sid1-mum3tassels initiated fewer BMs (Fig. 3H), and only four rows of SPM

3015RESEARCH REPORTids1 and sid1 regulate meristem fates

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 wasused as the outgroup. (C) Genomic structure of the maize sid1 gene. Boxes represent exons, dark boxes represent the AP2 domain, and trianglesrepresent 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 positionwithin the microRNA binding site over the total number of clones analyzed is indicated; those in parentheses to the right indicate the number ofclones 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 the28S 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 codingregion for the AP2 domain were used as probes.

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(Fig. 3I). The SM of the tassel in the double mutant wasindeterminate and continuously initiated lemma-like bracts withno FMs (Fig. 3J). The ear of the double mutant also initiated fewerrows (Fig. 3K, Fig. 4G). In contrast to the tassel, the SM of the earterminated in an ovule-like structure after initiating several bracts(Fig. 3L). No stamens or lodicules were observed, indicating thatthe normal pattern of floral organ initiation is not followed in thedouble mutant. Later in development, the tips of the bracts beganto differentiate structures that resemble silks (Fig. 3M).

Expression of sid1 in the wild type and ids1 andts4 mutantsTo examine the expression of sid1 more precisely, in situhybridization on wild-type and mutant tissue was performed. sid1transcript 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 floralorgans, 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 extrameristems. The ts4 ear tip showed ectopic sid1 expression near theinflorescence meristem tip, as well as higher-level expression in thelateral meristems (Fig. 4E).

Expression patterns of meristem markers suggestthat the SM never transitions to FM fate inids1;sid1 mutantsIn situ hybridization using meristem-specific markers was carriedout to further understand the basis for the indeterminacy and organidentity defects in ids1;sid1 double mutants. The maize knotted1(kn1) gene is expressed in the indeterminate cells of the meristemand is downregulated upon organ initiation (Jackson et al., 1994).Cross-sections of ids1-Burr ears compared with ids1-Burr;sid1-mum3 ears showed that the number of rows of SPMs was reduced(Fig. 4, compare F with G). kn1 expression in the spikelets of thedouble mutant persisted after the initiation of several sterile bracts(Fig. 4H), indicating that the meristem is indeterminate. Thebranched silkless1 (bd1) gene is expressed in a semi-circular domainat the base of the SM and disappears upon floret initiation in maizeand rice (Chuck et al., 2002). In young ids1-Burr;sid1-mum3spikelets, the bd1 expression pattern appeared normal (Fig. 4I).Later in development, however, instead of disappearing, thisexpression pattern persisted (Fig. 4J), demonstrating that themeristem had retained SM identity longer than normal.

The maize AGAMOUS-like MADS-box genes zmm2 and zag1 areduplicate genes that mark the FM as well as stamens and carpels(Schmidt et al., 1993; Mena et al., 1996) (Fig. 4K,M). They are notexpressed in leaf-like organs, such as palea or lemma. zag1mutations cause extra carpels to form that fail to fuse into afunctional silk (Mena et al., 1996). At early stages of spikeletdevelopment, we saw no expression of zmm2 (data not shown) orzag1 (Fig. 4N, inset) in the ids1;sid1 double mutant. However,ectopic zmm2 expression was observed later in the SM of the doublemutant as well as in the carpelloid bracts near the apex (Fig. 4L).zag1 was ectopically expressed in these same regions (Fig. 4N). Theectopic expression of AGAMOUS-like MADS-box genes in theids1;sid1 double mutant is consistent with the role of theArabidopsis AP2 gene as a negative regulator of AGAMOUS (Drewset al., 1991). Finally, the maize AP3 ortholog, silky1, which isnecessary 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 lodiculesof wild-type controls (Fig. 4O).

Based on previously described differences between the ts4 andTs6 mutant phenotypes (Chuck et al., 2007), it was postulated that atleast one other gene was targeted by the ts4 microRNA in additionto ids1. sid1 appears to be that gene based on four criteria: itsexpression levels are elevated in ts4 mutants (Fig. 1D,F,G); itscleavage is reduced in a ts4 mutant background (Fig. 1E); it isectopically expressed in ts4 mutant inflorescences (Fig. 4E); andsid1 mutants enhance suppression of the ts4 mutant phenotype byids1-mum1 (Fig. 2N). Previous work showed that MIR172 represses

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Fig. 2. sid1/ids1 mutant phenotypes. (A) Tassels of maize ids1-Burr(left), sid1-mum3;ids1-Burr (middle) and ids1-mum1;sid1-mum1;ts4-Atriple (right) mutants. (B) Mature ear of sid1-mum3;ids1-Burr. Barerachis is present in place of missing rows. (C) Fertilized ears of sid1-mum3;ids1-Burr (left) compared with ids1-Burr (right). (D) Hand sectionof wild-type (WT) ear spikelet. (E) Hand section of ids1-mum1 earspikelet. Extra florets are arranged laterally in the axils of bracts. (F) Hand section of sid1-mum3;ids1-Burr ear spikelet. Floret-likestructure is terminal. (G) Isolated bract surrounding ovule of sid1-mum3;ids1-Burr ear spikelet showing silk transformation at tip. (H) Wild-type tassel spikelet showing two florets (arrowheads). (I) ids1-mum1 tassel spikelet with three florets (arrowheads). (J) sid1-mum3;ids1-Burr tassel spikelet showing absence of florets. (K) ts4-A;ids1-Burr double mutant tassel (left) and ear (right). The tip of the earis highly branched. (L) Tassel of ts4-A;sid1-mum1 double mutant. (M) Tassel of plant that is homozygous for ts4-A and sid1-mum1 butheterozygous for ids1-mum1. (N) Ear of ts4-A;sid1-mum1 doublemutant (left) and of ts4-A;sid1-mum1 double mutant heterozygous forids1-mum1 (right). Plants are siblings.

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its AP2 target genes at the level of translation (Chen, 2004;Aukerman and Sakai, 2003). Other groups have found that MIR172repression may act at both the level of translation and transcriptcleavage (Schwab et al., 2005). We showed that IDS1 is regulatedat the level of translation by ts4 (Chuck et al., 2007). Our analysis ofsid1 suggests that it is regulated at the level of transcript stability,although we cannot rule out an additional level of translationalregulation. Thus, it is likely that ts4 regulates AP2 transcripts usingboth modes of regulation.

Lack of negative regulation of ids1 in Ts6 mutants leads to SMsthat initiate extra florets in a random phyllotaxy (Irish, 1997; Chucket al., 2007). Surprisingly, ids1 mutants also initiate extra florets, butin a defined distichous phyllotaxy. Thus, the loss-of-function andgain-of-function phenotypes for ids1 seem similar with regard tofloret initiation. This conflicting result can be explained by thefunction of its redundant paralog, sid1, which masks the ids1 loss-of-function null phenotype. Loss of both sid1 and ids1 leads to lossof FM initiation in the tassel and ear. Therefore, the gain-of-functionphenotype of ids1 (Ts6) shows that it is sufficient for FM initiation,and the complete loss of function of ids1 and sid1 shows that it isnecessary for FM initiation.

Loss of ids1 and sid1 also results in a reduction in the number oflateral meristems that initiate from the inflorescence meristem. Thisresult 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 formsof 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 theinflorescence meristems of the wild type and ids1;sid1 mutant (Fig.4A,H) showed that they were approximately the same size. Thisindicates that the more likely function of ids1 and sid1 is to regulateinitiation of lateral meristems in the inflorescence, rather thanregulating stem cell maintenance.

The carpelloid bract defects in ids1-Burr;sid1-mum3 ear spikeletsappear to be caused by ectopic expression of AGAMOUS-likeMADS-box genes in the lateral organs of the spikelet. AP2 mutantsin Arabidopsis display carpelloid sepals owing to ectopic expressionof AGAMOUS in those organs (Drews et al., 1991). The ids1;sid1floral organ phenotype demonstrates that this property is conservedin maize. Interestingly, the ectopic in situ expression of zag1 andzmm2 was not observed in the tassel (data not shown), which mightindicate that these genes have divergent functions in the male andfemale inflorescences, as previously suggested (Mena et al., 1996).

The ids1;sid1 tassel does not initiate FMs or floral organs. Thisfunction is unique to maize, because ap2 mutants in Arabidopsiscontinue to make flowers. A role for ap2 in FM fate is seen in doublemutants with ap1. Arabidopsis ap2;ap1 mutant flowers are less

3017RESEARCH REPORTids1 and sid1 regulate meristem fates

Fig. 3. Scanning electron microscopy of wildtype, ids1-mum1 and ids1-mum1;sid1-mum3. (A) Wild-type maize tassel, side view.(B) Wild-type tassel, top view, showing sevenrows of SPM (arrowheads). (C) Wild-type earfloret with initiating floral organs. Carpels aregrowing over the ovule primordium. (D) Olderear floret showing growing silk composed offused carpels. (E) Top view of ids1-Burr earshowing six SPM rows (arrowheads). (F) ids1-Burrspikelets with florets in the axils of extra lemmabracts. (G) Older ids1-Burr spikelet with initiatingfloral organs. Floral organs initiate in a patternsimilar to wild type. (H-M) ids1-Burr;sid1-mum4double mutant. (H) Tassel primordium with asingle BM (arrowhead). (I) Top view of tasselshowing four rows of SPM (arrowheads). (J)Indeterminate male spikelets with multiple bractsand no florets. (K) Young ear primordium withfour rows of SPM (arrowheads). (L) Femalespikelet terminating in an ovule primordium. (M) Older female spikelet with bract-to-silktransformations. 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. Scalebars: 100 μm.

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determinate, and initiate axillary structures in a helical pattern thatdevelop as pistils with occasional stamens (Irish and Sussex, 1990;Shannon and Meeks-Wagner, 1993). The lfy mutant of Arabidopsisdisplays striking similarities to maize ids1;sid1. Both lfy andids1;sid1 mutants are indeterminate, initiate several bracts beforeterminating in carpel-like structures (Huala and Sussex, 1992), andlack expression of B-function genes such as APETALA3/silky1(Weigel and Meyerowitz, 1993). The lfy phenotype is most obviouson early-formed flowers and, like ap2, is enhanced in doublemutants with ap1 (Huala and Sussex, 1992; Weigel et al., 1992;Shannon and Meeks-Wagner, 1993). Thus, LFY, AP1 and AP2 areall thought to play a role in the transition from IM to FM inArabidopsis.

The maize lfy double mutant, zfl1;zfl2, displays additionalsimilarities to ids1;sid1 in that fewer tassel branches form andstamen development is affected (Bomblies et al., 2003). Unlikeids1;sid1, however, zfl1;zfl2 mutants display indeterminacy in thecarpel and FM, and not in the SM. Moreover, the ids1;sid1phenotype is distinct in that both FMs and sex organs fail to form.The uniqueness of the ids1;sid1 phenotype might be due to itseffects on the fate of the SM, which normally initiates sterile bractsbefore florets are made. If ids1 and sid1 are required for the SM to

transition to FM initiation, then a block at this step could lead to theformation of multiple sterile organs. Since floral bracts are presentin both Arabidopsis and maize, these pathways are likely to beparallel in both monocots and dicots. One intriguing possibility isthat the divergence of monocots and dicots has led the AP2 genes tosubstitute for LFY function in the grasses. Analysis of sid1 and ids1in other genera closer to the monocot/dicot divide will elucidate theevolution 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 DevinO’Conner for phylogenetic analysis. This work was supported by NationalScience Foundation (NSF) grant DBI-0604923 and by USDA-ARS CurrentResearch Information System (CRIS) grant 5335-21000-018-00D to S.H., andby Cooperative State Research, Education and Extension Service (CSREES)grant 2004-35301-14507 to G.C.

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Fig. 4. In situ hybridization analysis of wild type, ts4and ids1;sid1 mutants. Probes are listed at lower left ofeach panel, genotypes at lower right. (A-E) sid1 in situs. (A) Wild-type maize tassel primordium. (B) sid1-mum2tassel primordium. (C) Wild-type ear florets with initiatingfloral organs. (D) ids-mum1 ear florets. (E) ts4-A earinflorescence. (F-H) kn1 in situs. (F) Cross-section of ear ofids1-Burr. Arrowheads indicate rows. (G) Cross-section ofear of ids1-Burr;sid1-mum3. (H) Close-up of spikelet ofdouble mutant showing prolonged kn1 expression. (I,J) bd1in situs. Expression marked with arrowheads. (I) Longitudinal section of ids1-Burr;sid1-mum3 ear showingnormal expression. (J) Older spikelets of double mutantshowing prolonged expression. (K,L) zmm2 in situs. (K) Wild-type ear florets with initiating floral organs. (L) ids1-Burr;sid1-mum3 ear spikelets with ectopicexpression 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 expressionin bracts and meristem. Inset shows a younger stage. (O,P) silky1 in situs. (O) Wild-type ear florets with initiatingfloral organs. (P) ids1-Burr;sid1-mum3 ear spikelets showingno expression. Lo, lodicules.

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3019RESEARCH REPORTids1 and sid1 regulate meristem fates

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