The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12688 This article is protected by copyright. All rights reserved.

Received Date : 26-Oct-2013 Revised Date : 12-Sep-2014 Accepted Date : 19-Sep-2014 Article type : Original Article The Polycomb Group Gene EMF2B is essential for maintenance of floral meristem determinacy

in rice

Liza J Conrad1, 2, Imtiyaz Khanday3, Cameron Johnson1, Emmanuel Guiderdoni4, Gynheung An5,

Usha Vijayraghavan3, Venkatesan Sundaresan1

1 Plant Biology Department, University of California Davis, Davis, CA

2 Current address: Biology Department, Eckerd College, St. Petersburg, FL

3 Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012,

India

4 CIRAD, UMR AGAP, TA 108/03 Avenue Agropolis, 34398 Montpellier Cedex 5, France

5 Department of Genetic Engineering and Crop Biotech Institute, Kyung Hee University, Yongin

446-701, Korea

Corresponding Author:

Venkatesan Sundaresan

University of California Davis

Plant Biology Department

1 Shields Ave

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This article is protected by copyright. All rights reserved.

Davis, California 95616

Phone: (530) 754 9677

Fax: (530) 752-5410

Email: [email protected]

Running Title: EMF2B promotes floral meristem determinacy in rice

Keywords: EMF2B, Polycomb, Floral Meristem, Oryza sativa, MADS-box

SUMMARY

The Polycomb Repressive Complex 2 (PRC2) represses transcriptional activity of target

genes through trimethylation of Lysine 27 of Histone H3. The functions of the plant PRC2 have

been chiefly described in Arabidopsis but specific functions in other plant species, especially the

cereals, are still largely unknown. Here we characterize mutants in the rice EMF2B gene, an

ortholog of the Arabidopsis EMBRYONIC FLOWER2 (EMF2) gene. Loss of EMF2B in rice

results in complete sterility, and mutant flowers have severe floral organ defects and

indeterminacy that resemble loss of function mutants in E-function floral organ specification

genes. Transcriptome analysis identified the E-function genes OsMADS1, OsMADS6 and

OsMADS34 as differentially expressed in the emf2b mutant compared to wild type. OsMADS1

and OsMADS6 known to be required for meristem determinacy in rice, have reduced expression

in the emf2b mutant, whereas OsMADS34 which interacts genetically with OsMADS1 was

ectopically expressed. Chromatin immunoprecipitation for H3K27me3 followed by qRT-PCR

showed that all three genes are presumptive targets of the PRC2 in the meristem. Therefore, in

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rice, and possibly other cereals, the PRC2 appears to play a major role in floral meristem

determinacy through modulation of the expression of E-function genes.

INTRODUCTION

First identified in Drosophila, Polycomb Group (PcG) proteins assemble into chromatin-

remodeling complexes to regulate genes in numerous developmental pathways in both plants and

animals (Duncan, 1982; Jurgens, 1985; Lewis, 1978); reviewed in (Francis and Kingston, 2001;

Pien and Grossniklaus, 2007; Schuettengruber and Cavalli, 2009). PcG complexes are well

conserved in molecular function and composition between Drosophila, mammals and plants

(Guitton and Berger, 2005). Two core Polycomb Repressive Complexes, PRC1 and PRC2, have

been identified. The PRC1 complex monoubiquitylates Lysine 119 of Histone H2A (reviewed in

(Guitton and Berger, 2005; Margueron and Reinberg, 2011) while the PRC2 is responsible for

tri-methylation of lysine 27 of Histone H3, the hallmark of PcG silenced chromatin (Cao et al.,

2002; Czermin et al., 2002; Ketel et al., 2005; Kuzmichev et al., 2002; Muller et al., 2002;

Nallamilli et al., 2013; Nekrasov et al., 2005).

Several genes with homology to the Drosophila PRC2 proteins have been identified in

Arabidopsis (reviewed in (Guitton and Berger, 2005; Pien and Grossniklaus, 2007). These

proteins function in at least three developmental stage specific PRC2 complexes to repress

homeotic genes controlling several transitions from the vegetative to reproductive phases in the

Arabidopsis life cycle (Guitton and Berger, 2005). In Arabidopsis, the PRC2 regulates flower

development through repression of the C function floral homeotic gene AGAMOUS (AG)

(Goodrich et al., 1997). Loss of either PRC2 component CURLY LEAF (CLF), or EMBRYONIC

FLOWER 2 (EMF2), causes ectopic expression of AG in both floral and vegetative tissues. This

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results in a mild floral phenotype with homeotic transformation of sepals and petals to carpelloid

and stamenoid organs, respectively (Goodrich et al., 1997; Moon et al., 2003; Yoshida et al.,

2001). However, this floral defect is secondary to stronger vegetative phenotypes in both

mutants; clf mutants possess leaves that are severely curled while emf2 mutants flower early,

almost immediately upon germination (Goodrich et al., 1997; Kinoshita et al., 2001; Moon et al.,

2003; Yang et al., 1995; Yoshida et al., 2001). Thus, the PRC2 containing CLF and EMF2 is

responsible for repression of AG in tissues outside of developing stamens and carpels, including

vegetative tissues. Significant functional redundancy exists between CLF and its paralog,

SWINGER (SWN). Single mutants of SWN show no floral phenotypes, but clf swn double

mutants exhibit floral phenotypes resembling those of Arabidopsis emf2 mutants; however in all

cases, the mutants continue to make determinate flowers (Chanvivattana et al., 2004). This is

consistent with the repression of AG by the wild-type PRC2, because AG promotes floral

determinacy in Arabidopsis through its target gene, KNUCKLES (KNU), which acts to repress

the meristem gene WUSCHEL (WUS) (Payne et al., 2004; Sun et al., 2009). However, more

recently it has been shown that mutants for PRC2 in Arabidopsis enhance the indeterminacy of a

weak allele of AG, revealing a secondary pathway to repress WUS through a direct interaction of

PRC2 with AG (Liu et al., 2011).

Two paralogs of EMF2 are present in rice, EMF2A (Os04g08034) and EMF2B

(Os09g13630). Both EMF2A and EMF2B expression is detected throughout the rice plants (Luo

et al., 2009). EMF2B has been shown to function to control flowering time in rice (Luo et al.,

2009; Yang et al., 2012) while no functional analysis or mutants have been reported for EMF2A.

These studies have reported abnormal floral organs and floret development in emf2b mutants

(Luo et al., 2009; Yang et al., 2012). However, the underlying causes of these floral phenotypes

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are not known, and meristem determinacy defects in these mutants have not been described.

Recently a mutation in the rice FIE1 gene has been shown to result in severe floral defects

(Zhang et al., 2012). The fie1 mutant was found to be an apparent gain-of-function epi-allele

that shows decreased methylation at the FIE1 locus; however, the question of meristem

determinacy was not addressed in that study.

Here, we have investigated the floral developmental functions of the PRC2 containing

EMF2B in rice. A detailed phenotypic characterization revealed that the loss of EMF2B results in

defects in all floral organs, as well as loss of determinacy of the floral meristem. From

transcriptome, chromatin immunoprecipitation, and in situ hybridization studies, we conclude

that in rice, EMF2B is necessary for the normal expression of E function genes required for both

floral organ specification and the establishment of floral meristem determinacy.

RESULTS

Loss of EMF2B results in defective floral organs and loss of determinacy

Three independent mutant alleles of EMF2B were characterized. Two are T-DNA

insertions previously reported by Luo et al (2009), one in intron 19 (Postech: 3A-08856;(Jeong et

al., 2002), emf2b-1, and the other in intron 10 (Oryza Tag Line Génoplante: AERF09), emf2b-2.

The third allele, emf2b-3, was generated as part of this study through TILLING, and is a guanine

to thymine transversion of the last basepair of exon 14 (Figure S1). Loss of this splice-site is

predicted to cause a truncation due to an in-frame stop codon 24 basepairs downstream in intron

15, and behaves as a null allele. The emf2b-1 and emf2b-2 insertion alleles are also predicted to

be null alleles; this was confirmed for emf2b-1 (Figure S2). All three emf2b mutant alleles are

phenotypically very similar, giving rise to mutant homozygotes that are severely stunted and

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completely sterile (Figure 1A). As previously reported by (Luo et al., 2009), most panicles do

not elongate outside of the sheath and the ones that emerge have a reduced florets compared to

wild type (Figure 1B, C). In both emf2b-3 plants observed, panicles consisting of only a large

mass of carpels and complete absence of branches were observed (Figure 1D).

Wild type rice flowers contain four types of organs arising from the floral meristem. The

outermost organs consist of two bract-like structures called palea and lemma (Figure 2A) formed

in an alternate pattern. Inner to the palea and opposite to it are two lodicules. The third set of

organs are 6 stamens in a whorl and the fourth whorl contains one carpel arising laterally to the

floral meristem that terminates (Figure 2F) (Itoh et al., 2005; Yamaki et al., 2011). Loss of

EMF2B results in severe defects in all floral organs. The palea and lemma over proliferate

resulting in uneven growth or multiple nested palea/lemma-like organs (Figure 2 B-E). For closer

examination, 24 flowers were harvested randomly from six different plants representing all three

alleles including three emf2b-1 plants, two emf2b-2 and one emf2b-3 plant. In 10 out of 24

flowers, the palea and lemma did not completely enclose the flower due to an over-proliferation

of inner organs (Figure 2C). In order to clearly visualize the inner whorls the palea and lemma

were removed (Figure 2 F-J). Frequently (18/24) in the second whorl the lodicules are converted

to leaf-like structures or are not present at all (Figure 2H, I, J arrows). In one flower, ectopic

lodicules were observed (Figure 2G, arrow). There was a reduced number or complete loss of

stamens (18/24) (Figure 2 G-J). Typically any stamens that were formed were defective as they

do not fully mature or produce pollen (8/24) (Figure 2J). Most strikingly, the majority of flowers

(19/24) displayed floral indeterminacy. In the majority of emf2b flowers (18/24), multiple fused

carpels are present in the inner most regions of the flower (Figure 2I, J). Other types of

indeterminacy were also observed, such as extra palea or lemma (4/24) (Figure 2E, G), and

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occasionally (3/24), an enlarged carpel with multiple stigma (Figure S3). Indeterminate flowers

were observed in every homozygous mutant plant for all three mutant alleles. In summary, loss

of EMF2B results in incorrect floral organ specification and floral meristem indeterminacy.

Intriguingly, these floral phenotypes of the emf2b mutants resemble those described in the double

mutants for two E-function floral development genes, OsMADS1/LHS1 and OsMADS6/MFO1

(Li et al., 2010; Ohmori et al., 2009).

Spatial and temporal expression of EMF2B

Floral organs of rice arise from the floral meristem (FM) in sequential order starting from

the outer most organs, the lemma, during inflorescence stage 7 (In7) (Itoh et al., 2005). The

lemma primordium arises at stage Spikelet 3 (SP3) followed by the palea primordium at stage

SP4. Two lodicule primordia are formed on the lemma side at SP5. Next, six stamen primordia

form in a concentric whorl (SP6). At stage SP7, the stamen primordia differentiate into anthers

and filaments while a single carpel primordium arises on the lemma side of the floral meristem.

The floral meristem is terminated at SP8 when it is converted to an ovule and male and female

gametophytes are formed in the ovules and anthers. Given the strong floral phenotype observed

in emf2b, we examined the expression pattern of EMF2B during these developmental stages of

the panicle using in situ hybridization. EMF2B mRNA was previously reported to be present in

several tissues, using RT PCR including panicle, ovary, anther, 2 day old seeds, 4 day old

endosperm, roots and young leaves (Luo et al., 2009). EMF2B mRNA is present as early as In5

stage of panicle development in the rachis branch meristem (rbm) and primary branch meristems

(pbm) (Figure 3A). Subsequently during In7, EMF2B mRNA can be detected in the spikelet

meristems (SM) (Figure 3A) and remains in the floral meristem throughout development (Figure

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3B, C). Early in floral development, (stage SP4) EMF2B is present in the palea and lemma

primordia (Figure 3C). At SP7, EMF2B localizes to the stamen primordia with reduced but

detectable expression in the developing carpel (Figure 3D, E). Later in mature rice flowers

(SP8), EMF2B is mainly restricted to the anthers and continued weak expression in the carpel

(Figure 3F). To summarize, although EMF2B mRNA is present throughout the rice plant,

expression of EMF2b is strongest in the meristematic domains, which show developmental

differences in the stages examined.

Transcriptome analysis of wild type versus emf2b developing panicles

In order to identify specific genes that might underlie the floral phenotype of the emf2b

mutant, transcriptome analysis for differential expression was performed on developing wild

type and emf2b mutant panicles. Immature panicles ranging in size from 0.3-1 cm (stage In7)

were used for two biological replicates each from both wild type and the emf2b-1 mutant allele.

This stage is at the time of floral organ differentiation thus maximizing the number of floral

organ identity genes detectable. RNAseq resulted in a total of 230,171,608 sequencing reads of

which 189,156,382 could be mapped to the rice genome. Of these, 87,329,380 reads could be

uniquely assigned to exon models of 45,027 genes from the total of the 55,801 annotated

“LOC_” loci in the rice genome (RGAP 7.0). Among this subset were a total of 7563

differentially expressed genes (Appendix S1), of which the majority, 5485 out of 7563,

significantly differentially expressed genes are up regulated in the mutant compared to wild type

(Figure S4). This global loss of repression is predicted in a PRC2 mutant such as emf2b, because

PRC2 typically acts on a very large number of genes in a given genome (Bouyer et al., 2011;

Makarevitch et al., 2013; Weinhofer et al., 2010).

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Gene Ontology Analysis revealed a strong enrichment for transcription factor activity

(Figure S5), with 422 of the differentially expressed genes being assigned a molecular function

as a transcription factor (GO:0003700) (Appendix S2).

MADS-box transcription factors are known regulators of all aspects in plant

development, but especially floral organ identity and floral meristem determinacy. Fifteen

MADS-box transcription factor genes are significantly differentially expressed in the emf2b

mutant (Appendix S2). Ten of these MADS-box genes have been reported to function in floral

organ specification and/or floral determinacy in rice (Table 1) (reviewed in (Yoshida and

Nagato, 2011). Four of these genes, OsMADS21, OsMADS22, OsMADS34/PAP2, and

OsMADS47 are up-regulated in the mutant while six, OsMADS1/LHS1, OsMADS4/PI,

OsMADS6/MFO1, OsMADS8, OsMADS16/SPW1, and OsMADS55 are down regulated in emf2b

(Table 1). Additionally, two floral organ identity genes, OPEN BEAK (OPB) and DROOPING

LEAF (DL), are significantly down regulated in the emf2b mutant. Many of these genes have

specific functions in floral organ identity however only the E function genes OsMADS34,

OsMADS1, OsMADS6 and OsMADS8 are known to regulate floral meristem determinacy.

Loss of EMF2B affects E-function gene expression in the meristem

The E-function in rice is controlled by two gene families, SEPALLATA (SEP) and a sister

clade to SEP, the AGAMOUS-LIKE6 (AGL6) family. There are 5 SEP genes, OsMADS1,

OsMADS5, OsMADS7, OsMADS8, and OsMADS34, and two AGL6-like genes, OsMADS6 and

OsMADS17 (reviewed in (Yoshida and Nagato, 2011). As described above, three of the five rice

SEPALLATA (SEP) genes (OsMADS1, OsMADS8, OsMADS34), and OsMADS6, an ortholog of

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Arabidopsis AGL6, are differentially expressed in the emf2b mutant (Table 1). These rice E

function genes (SEP-like and AGL6-like) function across all floral whorls, also act in floral

meristem determinacy and show genetic and physical interactions (reviewed in (Yoshida and

Nagato, 2011) and (Kater et al., 2006).

OsMADS8 shows complete functional redundancy with a duplicate gene, OsMADS7 (Cui

et al., 2010). Since OsMADS7 is not differentially expressed in the emf2b mutant, the reduction

in OsMADS8 expression is likely not contributing to the emf2b mutant phenotype. The

expression of OsMADS1, OsMADS6 and OsMADS34 was validated by Quantitative Real Time

PCR using the same samples as the transcriptome profiling (Figure S6), verifying the overall

down-regulation of OsMADS1 and OsMADS6 in the emf2b mutant. However, the increased

expression of OsMADS34 in the mutant was not statistically significant using this method,

perhaps reflecting the lower sensitivity compared to RNAseq which has a lower background.

OsMADS1 is essential for the development of rice floret organs and for the floret

meristem termination, once all the organs are specified (Agrawal et al., 2005; Jeon et al., 2000;

Prasad et al., 2005). Therefore, we used RNA in situ hybridization to investigate potential

changes to the domain of expression of OsMADS1 in emf2b florets. OsMADS1 mRNA is first

detected in the incipient floret meristem in both wild-type and emf2b plants during spikelet

stages SP4 and SP4 (Figure 4A, F). Later in development (SP4) its expression becomes localized

to the lemma and palea primordia (Figure 4B) and the same expression pattern is followed in

emf2b florets as well (Figure 4G). OsMADS1 continues to express in the floret meristem center

through SP4-SP7, in addition to lemma and palea in the wild-type florets (Figure 4B, C, arrows;

(Prasad et al., 2005)). However, at these stages the expression in the FM center of emf2b florets

is not detectable (Figure 4G, H, arrows). Because OsMADS1 promotes floral determinacy, this

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reduction in expression could contribute to the floral indeterminacy observed in emf2b plants. In

wild-type mature florets (SP8), OsMADS1 expression is detectable in the lemma and palea

(Figure 4D, I). While there is no change in the spatial expression domain of OsMADS1, the

expression appears to be lower in emf2b florets as compared to wild-type (Figure 4C-4D and 4H-

4I).

OsMADS34 is upregulated in emf2b mutants, and has previously been shown to be

involved in floral organ specification together with OsMADS1 (Gao et al., 2010; Khanday et al.,

2013). Consequently, we investigated the expression pattern of OsMADS34 in developing wild

type and mutant panicles at stage In7. In situ hybridization of wild type panicles revealed that

OsMADS34 mRNA is present in the FM (Figure 5 A, B). In later stages, OsMADS34 mRNA is

present in the primordia of all four floral organs as they develop (Figure 5C). At maturity,

OsMADS34 mRNA localizes to internal portions (likely the vasculature) of the lemma and palea,

and to stamens and the carpel (Figure 5D). In wild type developing panicles, EMF2B and

OsMADS34 expression patterns overlap significantly although some differences are apparent.

For example, while high expression of OsMADS34 is detected in spikelet organs i.e.,

rudimentary glumes and empty glumes (Figure 5B), EMF2B is feebly expressed in these organs

(Figure 3B). Moreover, EMF2B is very strongly expressed in the stamen primordia at SP7

(Figure 3C, D) while lesser OsMADS34 mRNA is detected in this tissue at SP7 (Figure 5C).

Interestingly, later in development OsMADS34 mRNA becomes very localized to the developing

anthers in the stamens (Figure 5D) suggesting EMF2B could be involved in restricting or

regulating OsMADS34 expression in the stamens. Correspondingly the most consistent

phenotype observed in emf2b mutants is the lack of viable stamens.

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Loss of EMF2B results in a dramatic expansion of OsMADS34 expression throughout the

developing panicle and flower. Staging of emf2b mutant panicles relative to wild-type

development is challenging due to over-proliferation of floral organs and other defects (Figure 5

E-J). Nevertheless, mRNA can be strongly detected throughout the floral meristem, all organ

primordia and all organs at maturity (Figure 5 E-J). This suggests that EMF2B is responsible for

transcriptional repression of OsMADS34 through much of the developing rice panicle and

flowers. Interestingly in the mutant, OsMADS34 expression expands even beyond the domain

where EMF2B expression is observed. It is possible that EMF2B is expressed at levels

undetectable by in situ hybridization in these domains, and that very low levels of EMF2B are

sufficient for repression of OsMADS34. Another explanation could be that the PRC2 is required

to establish an initial silencing mark that then recruits other mechanisms of transcriptional

silencing including PRC1 (reviewed in (Bemer and Grossniklaus, 2012). Loss of PRC2 silencing

early during spikelet and floral meristem development may lead to a loss of further repression by

other mechanisms, such as Histone H3K4 methylation which is an activation mark, or possibly

OsMADS34 itself as part of a self-reinforcing loop. Thus, the PRC2 could initiate silencing of

OsMADS34 in spikelet meristems, with re-enforcement and the maintenance of silencing within

different cells of the developing floral meristem occurring through other mechanisms.

MADS-box transcription factors are presumptive targets of the PRC2 for H3K27me3

methylation

The PRC2 is the only protein complex currently known to be responsible for the

H3K27me3 chromatin modification in plants (reviewed by (Bemer and Grossniklaus, 2012). To

investigate the histone methylation status of the differentially expressed MADS-box genes,

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chromatin immunoprecipitation (ChIP) with an H3K27me3 antibody followed by qPCR was

performed on wild type leaf and panicle tissues. Eighty-five wild type plants were dissected in

order to collect 32 panicles of the correct stage (In7). These panicles were used for three

biological replicates of approximately 100 mg each, which is at the lower limit of material

(typically 1 - 5 g) recommended for ChIP studies. qPCR primers designed to the 5’ region of

each gene were used to assay for enrichment in the H3K27me3 immunoprecipitated sample (see

Methods). OsMADS1 and OsMADS6 were both significantly enriched for H3K27me3 in both

tissues compared to a negative control, ACTIN (Figure 6A, B). In leaf tissue, OsMADS34 was

enriched for H3K27me3, whereas by contrast, very little H3K27me3 is detected at OsMAD22 in

leaves (Fig. 6A). ChIP performed on wild type developing panicles at In7 showed that

OsMADS22 was highly enriched for H3K27me3, while OsMADS34 was enriched to a lesser

extent (Figure 6B). Unfortunately, the prohibitively small size of emf2b mutant panicles along

with their stunted development made it unfeasible to obtain sufficient material to carry out ChIP

studies with mutant panicles. Nevertheless, the results with wild type panicles are consistent with

OsMADS34 being active in the developing panicle and its role in proper panicle and floral

development. The transcriptome analysis confirms that OsMADS34 is expressed at these stages

in wild type plants, although not throughout the whole inflorescence (see above); thus the overall

reduction of H3K27me3 when compared to leaf could be due to the samples containing a

mixture of tissues and stages, including those at which OsMADS34 is expressed. In summary,

H3K27me3 profiling suggests that the E-function genes OsMADS1, OsMADS6 and OsMADS34

are targets of PRC2 regulation in the developing panicle.

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DISCUSSION

EMF2 is a C2H2 Zinc finger protein (Yoshida et al., 2001). The gene family in

Arabidopsis contains three paralogous genes, EMF2, VRN2 and FIS2 (Yoshida et al., 2001).

Phylogenetic analysis has shown that an EMF2 ancestor was present prior to the divergence of

plants and animals (Chen et al., 2009). Loss of EMF2 in Arabidopsis results in early flowering,

immediately upon germination. Additionally, strong emf2 mutants have a mild floral phenotype

where sepals and stamens are transformed into carpelloid and stamenoid organs, respectively.

Nevertheless, the Arabidopsis emf2 mutant flowers are determinate, as is also the case with

mutants in CLF, another component of PRC2 in Arabidopsis (Goodrich et al., 1997; Kinoshita et

al., 2001; Moon et al., 2003; Yang et al., 1995; Yoshida et al., 2001). We have shown here that

the rice gene EMF2B plays a major role in floral meristem determinacy, and more generally in

floral organ development. Mutant emf2b flowers are affected in all floral whorls and exhibit

indeterminacy in the floral meristem center. The effects of loss-of-function of EMF2B

phenocopies the loss of function of two rice E-function genes, OsMADS1 and OsMADS6 as

discussed below.

EMF2b is required for regulated expression of a subset of floral MADS-box genes

Eight genes known to be involved in floral organ specification are down regulated in the

emf2b mutant, OsMADS1, OsMADS4, OsMADS6, OsMADS8, OsMADS16, OsMADS55, DL, and

OPB (Table 1). This down-regulation is likely to have a phenotypic effect on emf2b flowers.

Lodicules are affected in osmads1, osmads6, and opb mutants by either a transformation to palea

or lemma like organs (Chen et al., 2006; Ohmori et al., 2009; Prasad et al., 2005; Xiao et al.,

2009) or a reduced number of lodicules (Li et al., 2010). Additionally, reduced stamen number is

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a common phenotype observed with loss of function mutations in OsMADS1 (Chen et al., 2009;

Prasad et al., 2005), OsMADS6 (Li et al., 2010; Ohmori et al., 2009), and OPB (Xiao et al.,

2009). Finally, multiple fused or ill-formed carpels are observed in mutants of OsMADS1 (Chen

et al., 2006; Prasad et al., 2005), OsMADS6 (Kobayashi et al., 2010; Li et al., 2010; Ohmori et

al., 2009), and OPB (Luo et al., 2006). Both OsMADS1 and OsMADS6 have been implicated in

floral meristem determinacy (Agrawal et al., 2005; Li et al., 2010; Ohmori et al., 2009). We note

that the maize bearded ear (bde) mutant, which is a loss-of-function mutation in the maize

ortholog of OsMADS6, also exhibits floral indeterminacy phenotypes, suggesting that this

function of MADS6 is likely conserved among the grasses (Thompson et al., 2009). Overall,

floral defects observed in the emf2b mutant most closely resemble single and double mutants for

OsMADS1 and OsMADS6. In particular, osmads1 osmads6 double mutants show an extensive

loss of meristem determinacy (Ohmori et al., 2009), including the striking phenotype of profuse

carpels that is also observed in emf2b plants (Figure 1D, 2J). To summarize, the effects on floral

development of the loss of EMF2B are consistent with a reduction of OsMADS1 and OsMADS6

expression.

From the RNAseq analysis, only two MADS-box genes were significantly up regulated

in emf2b, OsMADS22 and OsMADS34. Both genes are enriched for histone H3K27me3

methylation, although the level of H3K27me3 at OsMADS34 appears to be lower in early stages

of panicle development as compared to leaves. OsMADS22 is strongly enriched for H3K27me3

in immature panicles, consistent with silencing of OsMADS22 after the transition from

vegetative to inflorescence meristem (Sentoku et al., 2005). Loss-of-function mutants in

OsMADS22 have no phenotype (Lee et al., 2008), while over-expression leads to spikelet

meristem indeterminacy and weak floral organ defects affecting primarily the first floral organs

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(Sentoku et al., 2005). These phenotypic differences with emf2b mutants, together with the

absence of known genetic or physical interactions between OsMADS22 and OsMADS1 or

OsMADS6, make it unlikely that mis-regulation of OsMADS22 leads to the floral phenotypes

observed in the emf2b mutants.

In situ hybridization confirmed that OsMADS34 is transcriptionally active at the In7 stage

of inflorescence development in wild-type plants, which has been shown by (Gao et al., 2010) to

be the peak of OsMADS34 expression; however, even at this stage, the expression is clearly

localized to specific domains within the meristematic region (Fig 5A). By contrast, in the emf2b

mutants, OsMADS34 expression is almost ubiquitous in the developing panicle and florets in

stages In7 through SP8, confirming that EMF2B is required for restricted expression of

OsMADS34 in the inflorescence and floral meristems. OsMADS34 has been implicated in floral

organ specification through genetic interaction with OsMADS1, and the function of both genes is

required for development of normal floral organs (Gao et al., 2010). OsMADS34 expression is to

some extent complementary to OsMADS1 (Gao et al., 2010; Khanday et al., 2013). Moreover,

antagonistic interactions between OsMADS1 and OsMADS34 have been shown, in that

OsMADS1 can repress OsMADS34 expression (Khanday et al., 2013). Thus, OsMADS34 might

be functioning within a network of MADS-box transcription factors that can regulate both organ

specification and FM determinacy including co-operative as well as antagonistic interactions

with factors such as OsMADS1.

PRC2 regulation of floral meristem determinacy and expression of rice E-function genes

Complex relationships can exist between gene expression and epigenetic modifications.

For example, in rice (He et al., 2010) concluded that even in the presence of H3K27me3 a gene

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could be highly expressed, possibly due to a high level of activating H3K4me3. Additionally,

maize genes differentially expressed in an Enhancer of Zeste (mez2/mez3) double mutant

compared to wild type are not enriched for Mez2/Mez3 dependent H3K27me3 (Makarevitch et

al., 2013). The Chip-qPCR data suggests that OsMADS1, OsMADS6, OsMADS34 are all

potential targets for PRC2 regulation in the developing panicle. Due to the repressive nature of

the PRC2, direct targets are predicted to show increased expression in a loss of function mutant

such as emf2b. The significantly reduced expression of OsMADS1 and OsMADS6 in the emf2b

mutant (Table 1) suggests that OsMADS1 and OsMADS6 are indirect targets of the PRC2 in the

panicle, despite the presence of H3K27me3 methylation at these loci. It has been demonstrated

that several of these E-function proteins are capable of interacting with one another in yeast,

specifically OsMADS1, OsMADS6 and OsMADS8 (Moon et al., 1999; Zhang et al., 2012).

OsMADS8 is also capable of interacting with another down-regulated gene in emf2b, OsMADS16

(AP3) and OsMADS13 (Kater et al., 2006). Additionally, OsMADS34 has been demonstrated to

interact several floral specification genes including the rice orthologs of AP1, OsMADS14 and

OsMADS15 (Kobayashi et al., 2012). Hypothetically, deregulation of OsMADS34 in the emf2b

mutant could alter the expression of genetically interacting genes such as OsMADS1 also

involved in floral meristem determinacy. Furthermore, other factors, not yet characterized to

function in floral development, may also be PRC2 regulated and co-operate with OsMADS34.

Divergent mechanisms for regulation of FM determinacy in rice and Arabidopsis

In Arabidopsis, floral meristem determinacy is controlled by C- and E-function genes as

related to the canonical ABC model for floral development, with one C-function gene, AG, and

four functionally redundant E-function genes, SEP1-4. Both ag mutants and sep triple mutants

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result in indeterminate flowers with repeating floral organs (Bowman et al., 1991; Ditta et al.,

2004). The primary mechanism for FM determinacy in Arabidopsis is that AG, presumably

acting in concert with SEP1-4, promotes expression of its target gene KNU, which in turn acts to

repress WUS (reviewed in (Irish, 2010). Mutant analysis in rice has also implicated both C- and

E-function genes in establishing FM determinacy. Rice contains two C-function genes,

OsMADS3 and OsMADS58, which are presumptive orthologs of AG (Yoshida and Nagato,

2011). (Dreni et al., 2011) have demonstrated that plants mutant for both OsMADS3 and

OsMADS58 display over-proliferation of lodicules, consistent with loss of determinacy.

Mutations in several rice SEP and AGL6-like genes result in FM determinacy defects, including

proliferation of carpels (reviewed in (Yoshida and Nagato, 2011). Therefore, FM determinacy in

rice is also under the control of both C- and E-function genes.

In Arabidopsis, loss of the PRC2 function alone does not have an effect on FM

determinacy. However, in a weak ag-10 mutant background, the PRC2 mutant curly leaf was

found to enhance the level of FM indeterminacy (Liu et al., 2011). This finding revealed a

secondary pathway for FM determinacy, by which the PRC2 is recruited by the C-function gene,

AG, to silence WUS (Liu et al., 2011). Hence the Arabidopsis PRC2 has multiple roles in

regulating flower development, on the one hand acting as a repressor of AG, but also co-

operating with AG to promote meristem determinacy in a secondary pathway to KNU.

Arabidopsis PRC2 has been recently shown to also act on the E-function gene SEP3, however,

this function does not relate to FM determinacy but to flowering time (Lopez-Vernaza et al.,

2012). In contrast, we have shown using emf2b mutants that in rice, loss of PRC2 function has a

major effect on FM determinacy. Notably, expression of known C-function genes is not affected

in these PRC2 mutants, but the expression of E-function genes OsMADS1 and OsMADS6 is

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significantly reduced, and the phenotypes observed are similar to loss of function of these two E-

function genes. Thus, in both Arabidopsis and rice, the PRC2 is involved in floral meristem

determinacy. The Arabidopsis PRC2 does not appear to have an essential role for establishment

of determinacy, but acts in an alternative pathway in concert with the C-function gene, AG to

silence WUS. The rice PRC2 on the other hand plays a primary role in determinacy of the floral

meristem, consistent with regulation of a genetic network involving the E-function genes. We

conclude that although floral determinacy in both Arabidopsis and rice requires similar sets of

organ identity genes, rice has evolved a distinct mechanism from Arabidopsis in the utilization of

chromatin silencing by the PRC2 complex to achieve the determinate state of the FM, a

mechanism that might be applicable to other grasses as well.

EXPERIMENTAL PROCEDURES

Plant growth conditions and genotyping

All rice plants were grown in greenhouse conditions of 30oC daytime and 25oC nighttime

temperatures. Light regimes were dictated by natural light conditions. For all three mutant

alleles, leaf tissue was collected and DNA extracted according to (Xu et al., 2005). Standard

PCR conditions recommended for MyTaq Red Mix (Bioline) were followed and products were

visualized using typical agarose gel electrophoresis, unless otherwise noted. The following gene

specific primers flanking the emf2b-1 insertion were designed using the Primer3 program

(http://frodo.wi.mit.edu/), 3A-08856u3 5’-TTGCATCCGTTTGAACTCTG-3’ and 3A-08856d2

5’-GTGCTGACAATCATGGCATC-3’. The primer used to detect the T-DNA insertion was,

2715RB: 5’-TTGGGGTTTCTACAGGACGTAAC-3’(Jeong et al., 2002). emf2b-2 primers were

published in (Luo et al., 2009) as follows: LB French: 5’-CGCTCATGTGTTGAGCATAT-3’,

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OsEMF2b ft F: 5’-TCTTTTGGGGCAGAAGTCAT-3’, and OsEMF2b ft R: 5’-

CACACGCTAATGGTCTGCTC-3’. The single base pair change of emf2b-3 ablates an Msp1

restriction enzyme site. Thus, genomic DNA was amplified as described above with primers,

emf2b-3 F: 5’TTTTCAGCTTTTGGCTGAGG-3’ and emf2b-3 R: 5’-

CCCTCTGCACGTAATCGTCT-3’. The PCR reaction was then digested with the Msp1 for 1

hour at 37oC. Products were visualized on a 2.5% Agarose TAE gel with ethidium bromide.

DNA carrying the emf2b-3 mutation will remain uncut (254 bp) while wild type DNA is digested

into two smaller fragments (52 bp, 202 bp).

Transcriptome analysis

Inflorescence 7 stage, according to (Itoh et al., 2005) (0.3-1.5 cm) was dissected from

rice plants (wild type Dongjin or homozygous emf2b-1 mutant), and placed in liquid Nitrogen.

Frozen tissue was homogenized using the Mini Beadbeater (BioSpec Products, Inc.). RNA was

extracted with Trizol Reagent (Invitrogen) using the manufacturers recommended protocol. RNA

was further purified with the QIAGEN RNAeasy Plant Mini kit (QIAGEN) following the

manufacturers protocol for RNA cleanup including the optional DNase digestion. RNA was

quantified on the ND 1000 Nanodrop Spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA

was synthesized using the Ovation RNA-seq System (NuGEN) with a starting input of 10 ng of

RNA. RNAseq libraries were created with the Encore NGS Multi Library Prep I kit (NuGEN).

Forty and fifty cycles of single-end of Illumina ultra high throughput sequencing was performed

on an Illumina HiSeq 2500 (Illumina).

The resulting non-partitioned barcoded Illumina fastq files were modified using a custom

perl script to classify reads to one of four barcodes (ACCC, CGTA, GAGT, TTAG) by

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classifying to the nearest one of eight possible Golay barcodes (ACCC, CGTA, GAGT, TTAG,

AGGG, CCAT, GTCA, TATC). Final reads having 1 or more undefined bases per 10 nucleotides

and/or being 20nt or less were discarded. The processed fastq files were then mapped to the rice

genome RGAP version 7.0 using TopHat v2.0.10 with default parameters except that the

microexon-search option was used and minimum intron size lowered to 20 nt. The resulting

accepted_hit.bam files were processed in conjunction with the RGAP 7.0 gene models file

(all.gff3) using a custom perl script that only counted reads that mapped within exons of the

defined gene models. The resulting genome-unique reads count table was reduced to a single

isoform for each 'LOC_' gene to the one that had the greatest number of uniquely mapping reads

across the four libraries. This data was then analyzed for differentially expressed genes using the

R package DESeq2 version DESeq2_1.0.19 with use of the default pipeline defined by the

function 'DESeq' that uses the Wald test to determine differential expression. A 10% false

discovery rate (FDR) was used as a cutoff to define differentially expressed genes. Gene

Ontology Analysis was performed through the AgriGO website

(http://bioinfo.cau.edu.cn/agriGO/) (Du et al., 2010).

In situ hybridization

Gene specific regions of OsMADS34 and EMF2B for probe preparation were amplified

with primer combination OsMADS34 FP 5’-GTT TGT GTT GAT TTG CTG GTC TGC TAG

AG-3’, OsMADS34 RP 5’-CGA TCC GCT GAA GCA CCA CCT TG-3’ and EMF2B FP 5’-

ACG CTT CTG TTG ATC CTG CT-3’, EMF2B RP 5’-TAT GGC CAT CCG CTA GTA CC-

3’respctively and cloned in pGEM T-easy vector (Promega, USA). Digoxigenin labeled sense

and antisense probes were transcribed by T7 and SP6 RNA polymerases respectively. Panicles of

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0.2 to 0.5 cm size were fixed in FAA and embedded in wax. The wax embedded panicles were

cut into 8µm thick sections and cross-linked to poly-L-lysine coated slides. Hybridizations were

performed as described in (Prasad et al., 2005). Each in situ hybridization experiment was

conducted on ~20 different panicles from different plants. The in situ hybridizations were

repeated three times for EMF2B and OsMADS34 probes, and twice for the OsMADS1 probe.

Detection of hybridized probes was done with DIG Nucleic acid detection kit (Roche

Diagnostics, Mannheim, Germany).

Chromatin immunoprecipitation

Chromatin from approximately 1.6g of mature leaf tissue or 100 mg of immature panicle

stage In7 from wild-type plants was used for each immunoprecipitation with H3K27me3

antibody (Millipore 07-449). Leaf chromatin was also immunoprecipitated with a purified rabbit

IgG antibody (Millipore) as a negative control yielding 1.29, 1, and 2.4 total nanograms of DNA

from samples WT1, WT2 and WT3, respectively. Chromatin Immunoprecipitation was

performed essentially according to (Bowler et al., 2004) with the following exceptions. All three

extraction buffers contained 1mM PMSF and lacked protease inhibitors. The nuclei lysis buffer

was modified from (Saleh et al., 2008) with only 0.1% SDA and no protease inhibitors.

Chromatin was sonicated on the high setting of a Bioruptor UCD-200 (Diagenode) for 8 thirty-

second cycles to produce approximately 500 bp fragments. Immunoprecipitation was performed

using Pierce Protein A/G Magnetic Beads (Thermo Scientific). Elution and reverse crosslinking

of chromatin was performed simultaneously for 2 hours at 65oC. ChIP purified DNA was

amplified using the Sigma Genomeplex Whole Genome Amplification (WGA2) kit following

the manufacturer’s protocol (Sigma-Aldrich Co.).

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Quantification of ChIP enrichment was performed by Quantitative PCR with ChIP and

input (no IP) DNA samples. Primers were designed using the Beacon Designer software

(BioRad). The rice ACTIN gene (Os03g50885) was utilized as a negative control for H3K27me3

with the following primers: Actin ChIP qPCR Forward: 5’-

GCTGTTTGGATCTCAGGGTGGTTTC-3’ Actin ChIP qPCR Reverse: 5’-

GCCGACAATGCTGGGGAAGAC-3’. Primers used to assayed OsMADS1, OsMADS22 and

OsMADS34 are as follows: OsMADS1 qPCR For: 5’-

GTAGCCAAACCACACCACCATAAAG-3’ OsMADS1 qPCR Rev: 5’-

ATCTTCTTCCTCCTCCTCTCCTCTC-3’. OsMADS22 qPCR For4: 5’-

GCCTTTGCCAGTTGCGGTGTTG-3’ OsMADS22 qPCR Rev4: 5’-

GAGGGTGAGGTGAGGTGAGGTGAG-3’. OsMADS34 qPCR For: 5’-

GTTTGTGTTGATTTGCTGGTCTGCTAGAG-3’ OsMADS34 qPCR Rev: 5’-

CGATCCGCTGAAGCACCACCTTG-3’. OsMADS6 primers (MADS1506f and MADS1645r)

can be found in (Zhang et al., 2010). These primers amplified the following genomic DNA

fragments relative to the transcription start site: OsMADS1 -85 to +2, OsMADS22 -328 to -194,

OsMADS34 -91 to +34, and OsMADS6 +1395 to +1513 (intron 1). Fold enrichment was

calculated relative to ACTIN.

ACKNOWLEDGEMENTS

We thank Dr. Luca Comai (UC Davis) for contributing the TILLING allele (emf2b-3) and Dr.

Mathias Lorieux (IRD-CIAT) for AERF09 seed. We are tremendously grateful to Julie Pelletier

and John Harada for assistance with the chromatin immunoprecipitation experiments and

technical advice. We also thank Cassandra Ramos, Paul Tisher, and Eugene Laurie for technical

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assistance. This work was partially funded by the U.S. Department of Agriculture NIFA grant

#2008-35304-04581.

SUPPORTING INFORMATION

Figure S1. Mutant alleles of EMF2B.

Figure S2. RT PCR of emf2b-1.

Figure S3. Enlarged emf2b-1 mutant carpel with multiple fused stigma.

Figure S4. Genes significantly differentially expressed in the emf2b-1 mutant versus wild type.

Figure S5. Molecular function Gene Ontology Analysis of genes differentially expressed in

emf2b panicles versus wild type.

Figure S6. Quantitative Real Time PCR validation of RNAseq for OsMADS1, OsMADS6, and

OsMADS34 expression.

Appendix S1. Genes differentially expressed in emf2b mutant

Differentially expressed genes at inflorescence stage 7 in emf2b mutant versus wild type.

Appendix S2. Differentially expressed transcription factors in the emf2b mutant

Gene Ontology Analysis was performed on all differentially expressed genes.

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TABLES Table 1. Differentially expressed transcription factors involved in floral development in emf2b

Function Name ID Log2FC padj

A MADS14 Os03g54160 NS - MADS15 Os07g01820 NS - MADS18 Os07g41370 NS -

B MADS16 Os06g49840 -2.17 0.0109 MADS2 Os01g66030 NS - MADS4 Os05g34940 -1.79 0.0505

C MADS3 Os01g10504 NS - MADS58 Os05g11414 NS -

D MADS13 Os12g10540 NS - MADS21 Os01g66290 3.18 0.00103

E

MADS7 Os08g41950 NS - MADS8 Os09g32948 -3.72 4.64E-12 MADS34 Os03g54170 1.03 0.0959 MADS1 Os03g11614 -1.88 9.51E-08 MADS5 Os06g06750 NS - MADS6 Os02g45770 -3.06 8.19E-18 MADS17 Os04g49150 NS -

SVP MADS22 Os02g52340 1.58 0.00166 MADS47 Os03g08754 2.49 0.00170 MADS55 Os06g11330 -1.14 0.00658

OPB OPENBEAK Os01g03840 -2.15 5.75E-12 DL DROOPING LEAF Os03g11600 -1.69 0.000119

FIGURE LEGENDS

Figure 1. Plant and panicle phenotypes of emf2b mutant.

(A) emf2b mutant plants (left) are stunted compared to wild type at maturity (right). Comparison

of wild type (B) and emf2b (C, D) panicles demonstrated the reduced branching and sterility in

emf2b mutant panicles. A severe over-proliferation of carpels was occasionally observed. Images

show are from (A) emf2b-1 (left), wild type Dongjin (right); (B) wild type Dongjin, (C) emf2b-2,

and (D), emf2b-3. Scale bars = 1 cm.

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Figure 2. Floral phenotypes of the emf2b mutant.

emf2b mutant flowers are defective in all four types of floral organs. Palea and lemma over

proliferate (B-E) and often do not completely enclose the inner floral organs (C). Lodicules are

converted to leaf-like structures (H-J), are absent or appear ectopically outside of the second

whorl (G). Stamens are rarely present (G-J) and do not mature or produce pollen (J). emf2b

mutant flowers typically contain multiple fused carpels (I, J) and are indeterminate (F-G). Palea

and lemma were removed in images (F-J). (A) Wild type flower (B) emf2b-1 (C) emf2b-3 (D)

emf2b-2 (E) emf2b-3 (F) Wild type (G) emf2b-3 (H) emf2b-1 (I) emf2b-1 (J) emf2b-2. Scale bars

= 1 mm.

Figure 3. In situ hybridization of EMF2B mRNA in developing wild type rice flowers.

EMF2B mRNA is detected in the rbm, pbm and sbm at stage In5 (A) and the SM at In7 (B) in

the developing inflorescence. (C) At spikelet stage SP4, EMF2B is expressed in the FM as well

as palea and lemma primordia. (D) and (E) Spikelet stage SP7 showing EMF2B in stamen

primordia and at a lower level in the carpel. (F) At maturity (SP8), EMF2B expression is

restricted to the anthers with weak expression in the carpels (arrows). Sense probes in In5 (G)

and SP8 (H). pbm = primary branch meristem, sm = spikelet meristem, rbm = rachis branch

meristem, fm = floral meristem, sl = sterile lemma, rg = rudimentary glume, le = lemma, pa =

palea, lo = lodicule, st = stamen, ca = carpel. Scale bars = 100 μm (A, B, G) and 50μm (C-F, H).

Figure 4. In situ hybridization of OsMADS1 mRNA on wild type and emf2b-2 mutant flowers.

(A-E) Wild type developing flowers. (A) OsMADS1 mRNA is detected in the FM early in

development at stage SP3. (B, C) At SP4 and SP7 OsMADS1 can be detected in the lemma, palea

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and in the outer layers of the developing carpels. (D) At maturity (SP8), OsMADS1 is localized

to the lemma and palea. (F-J) emf2b-2 mutant flowers. OsMADS1 is not detectable in the FM of

emf2b-2 mutant flowers compared to wild type (arrows). (E) Sense probe on wild type. (J) Sense

probe on emf2b-2. rg = rudimentary glume, sl = sterile lemma, fm = floral meristem, pa = palea,

lo = lodicule, st = stamen, ca = carpel. Scale bars = 100μm.

Figure 5. In situ hybridization of OsMADS34 mRNA in wild type and emf2b-3 mutant

developing flowers.

(A-D, L) Wild type developing panicles and flowers. (A) and (B) OsMADS34 is present in the

FM at panicle stage In7. (C) At spikelet stage SP3, OsMADS34 is expressed in all floral organ

primordia. (D) At maturity (SP8) OsMADS34 mRNA localizes to the inner palea and lemma, and

to stamens and carpels. In the emf2b-3 mutant panicles (E-J, K), OsMADS34 expression expands

throughout the floral meristem, all organ primordia and all organs at maturity. (K) sense probe

on emf2b-3, (L) sense probe on wild type. fm = floral meristem, sl = sterile lemma, rg =

rudimentary glume, le = lemma, pa = palea, lo = lodicule, st = stamen, ca = carpel. Scale bars =

100μm.

Figure 6. ChIP qPCR of MADS-box transcription factors.

ChIP performed on leaf tissue (A) and developing panicles at stage In7 (B). OsMADS6 and

OsMADS1 are significantly enriched for H3K27me3 in both tissues. OsMADS34 is significantly

enriched for H3K27me3 in leaf tissue and to a much lesser extent in immature panicles. In

contrast, OsMADS22 is highly enriched for H3K27me3 in panicles (B) but almost no H3K27me3

is detected in immature panicles (A). Three wild type biological replicates are shown as WT1,

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WT2 and WT3. Genes are displayed on the x-axis. Fold enrichment of H3K27me3 compared to

input is shown on the y-axis. Fold enrichment is shown relative to ACTIN.

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Documents

The polycomb group gene EMF2B is essential for maintenance of floral meristem determinacy in rice