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Overexpression of AtBMI1C,a Polycomb Group Protein Gene,Accelerates Flowering in Arabidopsis

Wei Li1.,Zheng Wang1,2.,Jian Li1,2,Hongchun Yang2,Sujuan Cui1,Xiaoxue Wang2*,Ligeng Ma1*

1Hebei Key Laboratory of Molecular Cell Biology,College of Biological Sciences,Hebei Normal University,Shijiazhuang,Hebei,China,2National Institute of Biological Sciences,Beijing,China

Abstract

Polycomb group protein(PcG)-mediated gene silencing is emerging as an essential developmental regulatory mechanism in eukaryotic organisms.PcGs inactivate or maintain the silenced state of their target chromatin by forming complexes,including Polycomb Repressive Complex1(PRC1)and2(PRC2).Three PRC2complexes have been identified and characterized in Arabidopsis;of these,the EMF and VRN complexes suppress flowering by catalyzing the trimethylation of lysine27on histone H3of FLOWER LOCUS T(FT)and FLOWER LOCUS C(FLC).However,little is known about the role of PRC1in regulating the floral transition,although AtRING1A,AtRING1B,AtBMI1A,and AtBMI1B are believed to regulate shoot apical meristem and embryonic development as components of PRC1.Moreover,among the five RING finger PcGs in the Arabidopsis genome,four have been characterized.Here,we report that the fifth, AtBMI1C,is a novel,ubiquitously expressed nuclear PcG protein and part of PRC1,which is evolutionarily conserved with Psc and BMI1.Overexpression of AtBMI1C caused increased H2A monoubiquitination and flowering defects in Arabidopsis.Both the suppression of FLC and activation of FT were observed in AtBMI1C-overexpressing lines,resulting in early flowering.No change in the H3K27me3level in FLC chromatin was detected in an AtBMI1C-overexpressing line.

Our results suggest that AtBMI1C participates in flowering time control by regulating the expression of FLC;moreover, the repression of FLC by AtBMI1C is not due to the activity of PRC2.Instead,it is likely the result of PRC1activity,into which AtBMI1C is integrated.

Citation:Li W,Wang Z,Li J,Yang H,Cui S,et al.(2011)Overexpression of AtBMI1C,a Polycomb Group Protein Gene,Accelerates Flowering in Arabidopsis.PLoS ONE6(6):e21364.doi:10.1371/journal.pone.0021364

Editor:Ivan Baxter,United States Department of Agriculture,Agricultural Research Service,United States of America

Received December21,2010;Accepted May31,2011;Published June20,2011

Copyright:?2011Li et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:This work was supported by grants from the China MOST863Project(to LM,2008105)and the Hebei Province Key Laboratory Program(to LM,109960121D).The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:ligeng.ma@https://www.wendangku.net/doc/5f14591882.html,(LM);xiaoxuewang6@https://www.wendangku.net/doc/5f14591882.html,(XW)

.These authors contributed equally to this work.

Introduction

Polycomb group proteins(PcGs)were first identified in Drosophila melanogaster,which maintains the repressed state of homeotic(Hox)genes during embryogenesis via histone methyl-ation[1].The PcGs identified to date form at least two different complexes,Polycomb Repressive Complex1(PRC1)and2 (PRC2),which have repressive functions in flies,humans,and plants[2,3].In Drosophila,PRC2is composed of four core proteins,including Enhancer of zeste E(z),an H3K27methyl-transferase,Extra sex comb(Esc),Suppressor of zeste12 (Su[z]12),and p55.This complex increases trimethyl H3K27 levels,thereby silencing Hox gene expression[4]and providing a recruitment site for PRC1[5].The PRC1core in Drosophila is composed of Polycomb(Pc),Posterior sex combs(Psc),Ring or Sex comb extra(Sce),and Polyhomeotic(Ph)[6,7].The mammalian PRC1complex contains HPC,HPH,RING1A/B, and BMI1,which are homologs of fly Pc,Ph,Ring(or Sce),and Psc,respectively[2].Among the RING finger proteins,dRing/ Sce and human RING1B have been shown to act as an E3 ubiquitylation ligase that monoubiquitylates lysine119in histone H2A[8].The other two RING domain-containing proteins,ligase activity.All three RING domain-containing proteins are required for PcG-mediated silencing[9,10].

In Arabidopsis,there are twelve homologs of Drosophila PRC2 subunits,which form three distinct PRC2-like complexes: EMBRYONIC FLOWER(EMF),VERNALIZATION(VRN), and FERTILIZATION INDEPENDENT SEED(FIS).These complexes play important roles in multifaceted developmental processes,including the vegetative phase transition,gametogene-sis,embryogenesis and early seed development,and flowering time control[11].

The first PRC2-like complex to be identified and biochemically characterized in Arabidopsis was the FIS complex,which prevents endosperm and seed development in the absence of fertilization [12].The FIS complex,which is composed of MEDEA(MEA), FERTILIZATION INDEPENDENT ENDOSPERM(FIE), FIS2,and MULTICOPY SUPPRESSOR of IRA1(MSI1) [13,14],represses the expression of PHERES1(PHE1)to prevent the central cell from initiating endosperm development before pollination by trimethylating H3K27in PHE1chromatin[15]. Inactivation of the FIS complex by fertilization or the delivery of other unknown factors results in the release of PHE1and triggers endosperm development[16].

flowering by targeting different branches of genes for silencing [17,18].During vegetative development,the EMF complex suppresses precocious flowering and enables vegetative develop-ment by repressing the transcription of FT and of the flower MADS box genes AGAMOUS and AGAMOUS-LIKE19by mediating the deposition of H3K27me3at their chromatin [18,19,20].Late during vegetative development and after flowering,CLF in the EMF complex binds directly to the chromatin of the floral repressor FLC and its relatives MAF4and MAF5,leading to H3K27me3modification and the repression of FLC under warm conditions[19].These results imply that the EMF complex regulates flowering in Arabidopsis by repressing the expression of these flowering genes at different stages of development[19,20].

The VRN complex containing VRN2,CLF,FIE,and MSI1is another PRC2-like complex that controls flowering and enables Arabidopsis to flower after vernalization[11].Cold exposure or vernalization promotes the formation and targeting of the PHD-VRN complex to FLC chromatin.The PHD-VRN complex increases H3K27me3levels at FLC chromatin,leading to sustainable silencing of FLC[21].The repressed state of FLC, then,is maintained epigenetically during subsequent plant development until it is reset during embryogenesis[22]. Although several PRC2-like complexes that control Arabidopsis development have been reported,PRC1-like complexes were identified only recently,primarily because there is no homolog of Drosophila Pc in the Arabidopsis genome[5].The existence of PRC1 in Arabidopsis was proposed recently based on evidence showing that the plant chromodomain protein LIKE HETEROCHRO-MATIN1(LHP1)binds H3K27me3in vitro and colocalizes genome-wide with H3K27me3profiles in euchromatin to turn off gene expression[23].

In Arabidopsis,five RING domain-containing PcGs have been predicted[24],and the functions of two RING1homologs (AtRING1A and AtRING1B)were recently characterized[25]. Severe cotyledon,rosette leaf,shoot apical meristem(SAM),flower morphology,and floral organ identity defects were observed in Atring1a/Atring1b,implying that a loss of function of both AtRING1A/B perturbs cell-fate determination[25].KNOX gene release was detected in Atring1a/Atring1b leaves,but the level of H3K27trimethylation at KNOX genes was unchanged,indicating that the suppression of KNOX genes by AtRING1A/B is independent of the H3K27trimethylation activity of PRC2[25]. However,the biochemical function of AtRING1A/B in PcG-mediated KNOX gene silencing has not been determined.

There are three BMI1homologs in Arabidopsis:AtBMI1A (At2G30580),AtRING1B(AT1G06770),and AtBMI1C (AT3G23060)[24].AtBMI1A/B regulate plant embryonic and stem cell development by functioning as E3ubiquitin ligases and components of PRC1[26,27].Specifically,AtBMI1A/B mediate the ubiquitination of DREB2A in response to water stress,leading to degradation of the protein by the26S proteasome[26].In addition, AtBMI1A/B were recently identified as components of PRC1[27]. AtBMI1A/B mediate H2A monoubiquitination,and some stem cell regulator genes were found to be expressed ectopically in Atbmi1a/ Atbmi1b cotyledons,implying that AtBMI1A/B are involved in silencing stem cell regulators and sustaining the differentiated state of somatic cells[27].In comparison,the function of the third BMI1 homolog,AtBMI1C,is unknown.

Here we report that AtBMI1C is a component of a PRC1-like complex and that it exhibits H2A monoubiquitination activity. AtBMI1C overexpression causes early flowering in Arabidopsis via silencing of the flowering repressor FLC and by promoting expression of the flowering activator FT.

Results

Three AtBMI1s are the homologs of human BMI1,a key component of PRC1

The mammalian PRC1ubiquitin E3ligase complex consists of several PcGs,including three RING domain-containing proteins (RING1/RING1A,RING2/RING1B,and BMI1)[8].To characterize the functions of BMI1homologs in Arabidopsis,we screened a whole-genome Arabidopsis sequence database using the protein or RING-domain sequence of Psc from Drosophila and its human homolog,BMI1.The existence of three human

BMI1-like Figure1.Phylogenetic relationship and conservation of AtBMI1s.(A)Phylogenetic tree based on the full-length sequence of AtBMI1from Arabidopsis and its human and fly homologs.(B)Tree showing the phylogenetic relationships among RING domain-containing AtBMI1s from Arabidopsis and their human and fly homologs.(C)Primary sequence alignment of the RING domain from AtBMI1and its human and fly homologs. The GenBank accessions of the sequences are:NM_128610(At2g30580)for AtBMI1A,NM_202046(At1g06770)for AtBMI1B,AY099845(At3g23060) for AtBMI1C from Arabidopsis thaliana,NM_002931for hRING1,NM_007212for hRING2from Homo sapiens,and NM_079001for Psc from Drosophila

proteins in Arabidopsis ,designated AtBMI1A (At2G30580),AtB-MI1B (A1G06770),and AtBMI1C (At3G23060),was revealed (Figure 1A);notably,each of these proteins was previously identified as a homolog of BMI1[24,27].

The RING domain of the BMI1s is conserved between Arabidopsis and animals (Figure 1B and C).The evolutionary conservation of a protein sequence implies functional similarity among distinct organisms.Both the full-length protein and RING-domain sequences of AtBMI1A were closely related to AtBMI1B,but slightly far from AtBMI1C in terms of their evolution (Figure 1A and B),indicating that AtBMI1C may function differently in Arabidopsis development.To date,only roles for AtBMI1A/B have been documented;the function of AtBMI1C in plant development remains unknown [27].Thus,we focused on AtBMI1C in our subsequent experiments.

AtBMI1C is a ubiquitously expressed nuclear protein

The expression pattern and subcellular localization of AtBMI1C were examined to elucidate the biological functions of the protein.To determine the subcellular localization of AtBMI1C,a reporter gene (Yellow Fluorescence Protein [YFP ])was fused to the AtBMI1C

coding region under the control of the CAULIFLOWER MOSAIC VIRUS (CaMV )35S promoter to generate stable transgenic plants carrying p35S::AtBMI1C-YFP or p35S::YFP (control).YFP signals were detected in the nucleus and cytoplasm in the roots of p35S::YFP transgenic plants (Figure 2A);in comparison,YFP signals in the roots or petals of p35S::AtBMI1C-YFP transgenic plants were detected only in the nucleus (Figure 2B and C).Thus,AtBMI1C encodes a nuclear-localized protein.

The spatial expression pattern of AtBMI1C was examined in various tissues by semiquantitative RT-PCR.Our results indicate weak expression of AtBMI1C in the SAM,juvenile leaves,adult leaves,stems,and cauline leaves,and abundant expression in cotyledons,inflorescences,siliques,seedlings,and roots (Figure 2D).Thus,AtBMI1C may function in multiple tissues during development.

AtBMI1C is a component of PRC1with H2A monoubiquitination activity

AtBMI1C was localized in the nucleus (Figure 2B and C),similar to AtRING1A and AtRING1B [25];thus,AtBMI1C was colocalized with AtRING1A and AtRING1B.To address whether AtBMI1C is a component of PRC1,we first explored

the

Figure 2.Nuclear localization and expression pattern of AtBMI1C .(A)Images of roots from transgenic seedlings harboring YFP driven by the CaMV35S promoter.(B)Images of roots from transgenic seedlings harboring YFP-tagged AtBMI1C driven by the CaMV35S promoter.Scale bar (red),100m m.(C)Images of petals from transgenic plants harboring YFP-tagged AtBMI1C driven by the CaMV35S promoter.Scale bar (red),50m m.(D)The

interactions of AtBMI1C with AtRING1A and AtRING1B,which are known components of PRC1in Arabidopsis[25],using a yeast two-hybrid assay.AtBMI1C interacted physically with AtRING1A and AtRING1B in yeast(Figure3B);the N-terminal domain of AtBMI1C,including the conserved RING domain,was required for this interaction(data not shown).The interaction of AtBMI1C with AtRING1A and AtRING1B was further confirmed by a pull-down assay.Both AtRING1A and AtRING1B could be pulled down by AtBMI1C(Figure3C),suggesting that AtBMI1C and AtRING1are in the same PRC1

complex.

Figure3.Physical interactions between AtBMI1C and AtRING1A/B,and the detection of H2A monoubiquitination activity.(A)Yeast two-hybrid assay.Positive control:pGADT7-T+pGBKT7-53(encoding fusions between the GAL4DNA-BD and AD and murine p53and SV40large T-antigen,respectively).Negative control:pGADT7-T+pGBKT7-Lam(encoding a fusion of the DNA-BD with human lamin C;control for interactions between an unrelated protein and either the pGADT7-T control or AD/library plasmid).The indicated combinations of plasmids were co-transformed into the yeast reporter strain,and the interactions of AtBMI1C with AtRINGs were assessed by growth on plates lacking Leu,Trp,His,and adenine.(B) The interactions between AtBMI1C and AtRING1A/B were quantitatively evaluated based on the level of b-galactosidase activity.(C)Pull-down assay. Total protein was extracted from2g of eleven-day-old Myc-RING1A/ring1or Myc-RING1B/ring1plants,respectively.Each protein extract was divided in half and incubated with MBP-or MBP-GST-coated beads.The pulled down fractions were analyzed by Western blotting.(D)Western blot analysis of histone extracts of WT and35S::BMI3-YFP using anti-ubiquitin and-H3antibodies,respectively.Molecular weight(MW)markers(in kDa),

For a long time,scientists believed that there was no H2A monoubiquitination activity in plants [25].However,recent data suggest the existence of an AtBMI1A-and AtBMI1B-containing PRC1complex with H2A monoubiquitination activity [27].To determine whether AtBMI1C-containing PRC1possesses H2A monoubiquitination activity,we measured the monoubiquitinated H2A level in wild-type plants and an AtBMI1C-overexpressing line (Figure 3D).Anti-ubiquitin antibodies recognized two specific bands from nuclear histone extracts (Figure 3D);the upper band was recognized by anti-monoubiquitinated H2B (uH2B)antibodies as well (data not shown),suggesting that the upper band was uH2B,while the lower band was monoubiquitinated H2A (uH2A).Moreover,the level of H2A monoubiquitination activity was increased in the AtBMI1C-overexpressing line (Figure 3D).However,there was no obvious change in H2B monoubiquitination activity between the wild-type plants and AtBMI1C-overexpressing lines (Figure 3D).This result suggests that,like its homologs,the AtBMI1C-containing complex exhibits H2A monoubiquitination activity in Arabidopsis .

No loss-of-function T-DNA insertion mutant of AtBMI1C was isolated

To investigate the biological function of AtBMI1C in Arabidopsis ,T-DNA insertion mutants of AtBMI1C were ordered from the Arabidopsis Biological Resource Center (ABRC).A homozygous T-DNA insertion allele of AtBMI1C was identified (Atbmi1c-1,

SALK_148143),in which the T-DNA was inserted upstream of the start codon (Figure 4A).However,the T-DNA insertion in Atbmi1c-1did not abolish the expression of AtBMI1C (Figure 4B).Not surprisingly,no visible phenotype was detected among the homozygous mutant plants (data not shown).

Lack of obvious defects in down-regulated RNA interference (RNAi)lines of AtBMI1C

Because no T-DNA insertion mutant of AtBMI1C was identified,RNAi was used to explore the function of AtBMI1C.Three primer pairs were designed using an online program (https://www.wendangku.net/doc/5f14591882.html,/cgi-bin/mirnatools.pl?page =1),the resulting three constructs were delivered to wild-type plants,and the expression of AtBMI1C was analyzed in T1independent lines.Several transgenic lines (AtBMI1C-Rs)were identified,including AtBMI1C-R1,AtBMI1C-R4,and AtBMI1C-R9,in which the expression of AtBMI1C was down-regulated (Figure 4C).Defects,especially in flowering time,were monitored in the next generation;however,no visible phenotype was observed in AtBMI1C-R1,AtBMI1C-R4,and AtBMI1C-R9compared to control (AtBMI1C-R12)and wild-type plants (Figure 4D and E).

Because no T-DNA insertion mutant was available and because no visible phenotype was observed in our amiRNAi lines of AtBMI1C ,we next investigated the function of the gene by overexpressing it in Arabidopsis

.

Figure 4.Identification of the AtBMI1C mutant and characterization of artificial microRNAi lines.(A)Genomic architecture of AtBMI1C and position of the mutation in atbmi1c-1.The 59or 39UTR is represented by a gray bar.Exons are represented by black bars.Introns are represented by black lines.The T-DNA insertion in atbmi1c-1(SALK_148143)is located in the 59UTR of AtBMI1C .Scale bar,500bp.(B)Detection of AtBMI1C mRNA in a homozygous atbmi1c-1T-DNA insertion line by semiquantitative RT-PCR.Total RNA was extracted from the inflorescences of homozygous atbmi1c and wild-type plants.Semiquantitative RT-PCR was performed to amplify the full-length transcript using ACTIN2/7as an endogenous control.(C)Characterization of AtBMI1C mRNA abundance in AtBMI1C-Rs.Total RNA was extracted from the inflorescences of AtBMI1C-Rs and wild-type plants.Semiquantitative RT-PCR was conducted to amplify the full-length transcript using ACTIN2/7as an endogenous control.(D)Morphology of the AtBMI1C-Rs,in which AtBMI1C was down-regulated,compared to wild type and AtBMI1C-R12,an amiRNAi line in which the expression of AtBMI1C was almost the same as in wild type.(E)Flowering time in the AtBMI1C-Rs was the same as in wild type.Plants were grown under LD conditions.The

AtBMI1C overexpression confers an early flowering phenotype in Arabidopsis

To examine whether the overexpression of AtBMI1C affects plant growth and development,we generated p35S::AtBMI1C-YFP transgenic plants and monitored their expression of AtBMI1C by semiquantitative RT-PCR (Figure 5B).We also recorded the phenotypes of the transgenic lines.The p35S::AtBMI1C-YFP transgenic lines showed an early flowering phenotype (Figure 5A).Moreover,using p35S::AtBMI1C-YFP-27(35S-27)and p35S::AtBMI1C-YFP-14(35S-14)as representatives,the trans-genic lines were found to possess far fewer rosette leaves than wild type under both long-day (LD;16h of light/8h of dark)and short-day (SD;8h of light/16h of dark)conditions (Figure 5C and D and Table 1).In addition,fewer days were required for emergence of the first bud and for opening of the first flower in lines 35S-27and 35S-14compared to wild-type plants (Table 1).To exclude the effect of YFP on the function of AtBMI1C,a construct containing only p35S::AtBMI1C was transformed into wild-type plants.Flowering time was monitored in the T1generation and in a number of T2independent lines.Flowering time in the transgenic plants containing p35S::AtBMI1C coincided with that in transgenic plants harboring p35S::AtBMI1C-YFP ,implying that the early flowering phenotype of the p35S::AtBMI1C-YFP transgenic plants was due to the overexpression of AtBMI1C ,rather than the fusion of YFP to AtBMI1C (data not shown).Arabidopsis undergoes at least two phase transitions during its life cycle:a vegetative and a reproductive phase transition [28].The vegetative phase transition represents a shift from the juvenile vegetative phase to the adult vegetative phase,which is usually defined by the production of leaves with abaxial trichomes;in comparison,the dramatic vegetative to reproductive phase transition,or floral transition,is characterized by bolting,flowering,and setting seeds for the next generation [28].To determine the effects of the overexpression of AtBMI1C on vegetative and reproductive phase transitions in Arabidopsis ,we examined the juvenile and adult leaf number in transgenic lines after bolting.There was no significant difference in juvenile leaf number between line 35S-27or 35S-14and wild type;

however,

Figure 5.AtBMI1C overexpression driven by the 35S promoter accelerates flowering in Arabidopsis .(A)Morphology of AtBMI1C -overexpressing plants carrying p35S::AtBMI1C-YFP grown under LD conditions for 27days.The plants flowered earlier than wild type.A total of 30out of 198independent T1lines showed an early flowering phenotype and elevated AtBMI1C expression.A number of independent transgenic lines were chosen for the following investigation.Scale bar,2cm.(B)AtBMI1C expression in transgenic lines carrying p35S::AtBMI1C-YFP .AtBMI1C expression was measured by semiquantitative RT-PCR.ACTIN2/7was used as an internal control.(C)and (D)Determination of flowering time in AtBMI1C -overexpressing plants containing p35S::AtBMI1C-YFP grown under LD and SD conditions using two AtBMI1C overexpressor lines as representatives.The number of rosette leaves was determined after bolting.(E)Vegetative phase transition in AtBMI1C -overexpressing plants containing p35S::AtBMI1C-YFP grown under LD conditions.Juvenile,adult,rosette,and cauline leaves were counted after flowering.Juvenile and adult leaves were distinguished based on the presence of trichomes on their abaxial surface.doi:10.1371/journal.pone.0021364.g005

Table 1.Flowering time in p35S::AtBMI1C-YFP plants grown under LD conditions.

Genotype Days to the first visible bud Days to the first open flower Rosette leaf number n Wild type 26.0060.3832.8360.3813.1160.141835S -2718.4360.47**24.5760.31** 6.4360.14**1435S -1419.6160.31**25.7860.34**8.2260.10**1835S -9

18.6260.10**

25.6260.10**

6.5860.10**

26

Data are presented as the mean 6SD;

**represents a significant difference from wild type (t -test,p ,0.01);

Figure6.Tissue-specific AtBMI1C overexpression promotes flowering in Arabidopsis.(A)Morphology of transgenic plants carrying pAP1::AtBMI1C-GFP grown under LD conditions for28days.The plants showed an early flowering phenotype compared with wild type.A total of18 out of72independent T1lines showed an early flowering phenotype.A few lines were chosen for the following experiments.(B)and(C) Determination of flowering time in transgenic plants containing pAP1::AtBMI1C-GFP grown under LD and SD conditions using three AtBMI1C transgenic lines as representatives.The number of rosette leaves was determined after bolting.(D)Vegetative phase transition in transgenic plants containing pAP1::AtBMI1C-GFP grown under LD conditions.Juvenile,adult,rosette,and cauline leaves were counted after flowering.Juvenile and adult leaves were distinguished based on the presence of trichomes on their abaxial surface.(E)AtBMI1C expression in transgenic lines carrying pAP1::AtBMI1C-YFP.Total RNA was extracted from leaves of pAP1::AtBMI1C-GFP and wild-type plants.AtBMI1C expression was measured by semiquantitative RT-PCR using ACTIN2/7as an internal control.(F)Morphology of transgenic plants carrying pKNAT1::AtBMI1C-GFP grown under LD

the adult leaf number in35S-27and35S-14was dramatically reduced(1.07and1.43adult leaves,respectively)compared with wild type(4.50adult leaves)(Figure5E),suggesting that the overexpression of AtBMI1C affected only the floral transition and not the vegetative phase transition.

Tissue-specific AtBMI1C expression also produces an early flowering phenotype in Arabidopsis

CaMV35S is a constitutive promoter with activity in various cell types,tissues,and organs.To investigate the relationship between AtBMI1C expression and the phenotype of the transgenic plants, several tissue-specific promoters were selected to drive the expression of AtBMI1C,including the APETALA1(AP1)promoter, a floral primordium-,sepal-,and petal-specific promoter[29]. pAP1::AtBMI1C-GFP was generated and introduced to wild-type plants.A total of18out of72independent transgenic lines harboring pAP1::AtBMI1C-GFP exhibited an early flowering phenotype under LD and SD conditions(Figure6A-C and Table2).In the mean time, the level of AtBMI1C expression in the pAP1::AtBMI1C-GFP transgenic plants was measured by semiquantitative RT-PCR. Elevated AtBMI1C expression was detected in the early flowering transgenic plants(Figure6E),indicating that the alteration in flowering time was caused by the overexpression of AtBMI1C.

We further investigated the impact of the overexpression of AtBMI1C using the AP1promoter on vegetative and reproductive phase transitions in a pAP1::AtBMI1C-GFP-9transgenic line(A-9). Our results were similar to those obtained for lines35S-27and35S-14.The juvenile leaf number in A-9was almost the same as in wild type;however,the adult leaf number in A-9(1.83adult leaves)was lower than that in wild type(6.33adult leaves)(Figure6D).Thus, the AP1-driven overexpression of AtBMI1C affected only the floral transition,and not the vegetative phase transition.

FLC suppression and FT activation in the AtBMI1C-overexpressing lines

FLC is a central floral repressor that blocks the expression of floral activators such as FT and SUPPRESSOR OF OVEREX-PRESSION OF CO1(SOC1)to prevent the initiation of flowering during vegetative development.The down-regulation of FLC activates FT and SOC1and promotes flowering[30].

To explore the molecular mechanisms responsible for the change in flowering time in our AtBMI1C-overexpressing lines,FLC and FT expression was examined by quantitative RT-PCR. The expression of FLC in lines35S-14and35S-27was2.5times lower than that in wild type(Figure7A).As a result of the down-regulation of FLC,the expression of FT in lines35S-14and35S-27 was about five times higher than that in wild type(Figure7B). Similarly,the repression of FLC in lines A-9,A-13,and A-14was also observed(Figure7C).An increase in FT expression of3-4.5 times in lines A-9,A-13,and A-14was detected(Figure7D).These results suggest that AtBMI1C overexpression promotes flowering by repressing FLC expression and activating FT expression. Early flowering caused by AtBMI1C overexpression in the SAM and vascular companion cells

FLC is expressed mainly in the SAM and vascular companion cells[31].Our initial results indicated that AtBMI1C overexpres-sion repressed FLC and raised the expression of FT to promote flowering.Thus,we hypothesized that expressing AtBMI1C in specific tissues,including the SAM and vascular companion cells, would cause early flowering.To test this hypothesis,KNAT1,an SAM-specific promoter[32],and SUC2,a vascular companion cell-specific promoter[33],were selected to drive the tissue-specific expression of AtBMI1C.

Flowering time in T1and T2lines expressing pKNAT1::AtB-MI1C-GFP and pSUC2::AtBMI1C-GFP was examined.As expected, AtBMI1C overexpression in either the SAM or vascular companion cells caused early flowering.Lines K-32,K-58,and K-79containing pKNAT1::AtBMI1C-GFP flowered earlier(8.22,8.50,and8.63 rosette leaves)than wild type(12.65rosette leaves)(Figure6F and G and Table3).Similarly,lines SU-58and SU-142carrying pSUC2::AtBMI1C-GFP produced fewer rosette leaves(8.33and 8.12,respectively)compared to wild type(12.71leaves)(Figure6H and I and Table4).

H3K27me3and H3K4me3modifications at FLC chromatin were unchanged in our AtBMI1C overexpressor line Histone modification at FLC chromatin plays an essential role in regulating FLC[34,35,36].The H3K27me3or H3K4me3modifi-cation of FLC chromatin is related to its repression or activation, respectively[34].To investigate the biological mechanisms underly-ing the down-regulation of FLC in our AtBMI1C-overexpressing lines, we performed chromatin immunoprecipitation analysis to determine the level of H3K27and H3K4trimethylation at FLC chromatin in

showed an early flowering phenotype.(G)Determination of flowering time in transgenic plants containing pKNAT1::AtBMI1C-GFP grown under LD conditions.The number of rosette leaves was determined after bolting.(H)Morphology of transgenic plants carrying pSUC2::AtBMI1C-GFP grown under LD conditions for28days.The plants showed an early flowering phenotype compared with wild type.A total of24out of108independent T1 lines showed an early flowering phenotype.(I)Determination of flowering time in transgenic plants containing pSUC2::AtBMI1C-GFP grown under LD conditions.The number of rosette leaves was determined after bolting.

doi:10.1371/journal.pone.0021364.g006

Table2.Flowering time in pAP1::AtBMI1C-GFP plants grown under LD conditions.

Genotype Days to the first visible bud Days to the first open flower Rosette leaf number n

Wild type28.4860.2334.6760.3212.0060.1927

AP1-923.0060.39**31.0060.35**9.1160.20**19

AP1-1322.8760.16**31.0060.18**9.1060.09**20

AP1-1422.5460.21**30.0060.30**9.0060.13**24

Data are presented as the mean6SD;

**represents a significant difference from wild type(t-test,p,0.01);

AtBMI1C -overexpressing line 35S-9.There was no obvious difference in the level of H3K27me3or H3K4me3across FLC chromatin between wild type and line 35S-9(Figure 8).Discussion

The PcG proteins in PRC2complexes (e.g.,the EMF complex and VRN complex),which are involved in the switch from vegetative to reproductive development,have been documented,and they have been shown to be involved in the H3K27me3modification of FT and FLC chromatin [11].However,little is known about the impact of PRC1components on the regulation of

flowering,even though PRC1components AtRING1A/B and AtBMI1A/B play essential roles in the repression of plant developmental regulators in Arabidopsis [25,27].

AtBMI1C is involved in flowering time control in Arabidopsis

Genetic and molecular analyses have uncovered multiple signaling pathways that integrate environmental and developmen-tal cues to control flowering time in Arabidopsis ,including the autonomous,photoperiod,vernalization,gibberellin,and PAF1complex pathways [37].PRC2complexes are involved in cold-

Table 3.Flowering time in KNAT1::ATBMI1C-GFP plants grown under LD conditions.

Genotype Days to the first visible bud Days to the first open flower Rosette leaf number n Wild type 27.0060.3533.8360.2912.6560.2218K-3222.2860.34**28.7860.34**8.2260.25**18K-5823.5060.60**30.0860.58**8.5060.30**12K-79

20.4760.29**

27.1160.25**

8.6360.14**

19

Data are presented as the mean 6SD;

**represents a significant difference from wild type (t -test,p ,0.01);Figure 7.Expression of FLC and FT in AtBMI1C -overexpressing lines.(A)Expression of FLC in AtBMI1C -overexpressing lines harboring p35S::ATBMI1C-YFP as determined by quantitative real-time RT-PCR.(B)FT expression in AtBMI1C -overexpressing lines harboring p35S::AtBMI1C-YFP as determined by quantitative real-time RT-PCR.(C)FLC expression in transgenic lines harboring pAP1::AtBMI1C-GFP as determined by quantitative real-time RT-PCR.(D)FT expression in transgenic lines harboring pAP1::AtBMI1C-GFP as determined by quantitative RT-PCR.Total RNA was isolated from ten-day-old transgenic or wild-type seedlings.FLC or FT expression was measured by quantitative real-time RT-PCR using ACTIN2/7as an endogenous control.The values are the mean 6SD of three independent experiments.doi:10.1371/journal.pone.0021364.g007

induced flowering regulation[34,37];however,no PRC1 component has been shown to act as a floral transition regulator. Because no loss-of-function AtBMI1C mutant was available until now,and given that no visible phenotype was observed in our RNAi lines(Figure4),we generated several transgenic lines in which AtBMI1C was overexpressed(35S-AtBMI1C-YFP,AP1-AtBMI1C-GFP,KNAT1-AtBMI1C-GFP,and SUC1-AtBMI1C-GFP) in order to dissect the functions of AtBMI1C in Arabidopsis. Flowering was accelerated in all AtBMI1C overexpressors(Figures5 and6).FLC is a central repressor of flowering that integrates endogenous signals from the autonomous and vernalization pathways with environmental cues[37].The repression of FLC and activation of FT were detected in our AtBMI1C overexpression lines(Figure7).These results suggest that AtBMI1C is involved in flowering regulation.Further analysis using an AtBMI1C knock-out mutant will provide additional evidence for the functions of AtBMI1C.

AtBMI1C is a PRC1component,and the AtBMI1C-containing complex exhibits H2A monoubiquitination activity

AtBMI1C is a homolog of the RING-domain proteins AtBMI1A/B in Arabidopsis,Psc in Drosophila,and BMI1in humans (Figure1B and C).In this report,we found that AtBMI1C interacts physically with AtRING1A/B(Figure3B and C), indicating that AtBMI1C is a novel component of PRC1in Arabidopsis.

In animals,the repression of gene expression is maintained by PRC1,which monoubiquitinates H2A[8–10].However,H2A monoubiquitination was not detected in plants until a recent report showing that AtBMI1A/B are required for H2A mono-ubiquitination activity[27].AtBMI1A/B-mediated H2A mono-ubiquitination activity has been shown in vivo and in vitro,and defects in AtBMI1A/B have been shown to impair the production of uH2A[27].In the present study,we found that an increase in the level of AtBMI1C raised H2A monoubiquitination activity (Figure3D).Thus,our result supports the notion that AtBMI1-containing PRC1has H2A monoubiquitination activity in Arabidopsis.

AtBMI1C-containing PRC1suppresses FLC expression independent of H3K27methylation

PRC1controls gene expression by altering the level of H2A monoubiquitination[8–10],while PRC2methylates histone H3 at lysine27to silence gene expression[2–4].In the present study,we found that AtBMI1C overexpression accelerated flowering and repressed the expression of FLC(Figures5–7).In addition,the repression of FLC by AtBMI1C is independent of complex to suppress the expression of FLC and promote flowering in Arabidopsis.However,we cannot rule out the possibility that FLC is not a direct target of AtBMI1C-containing PRC1at this stage.Further study is needed to resolve this issue.

In conclusion,a novel PcG in PRC1,AtBMI1C,was characterized in this study.AtBMI1C is a universally expressed nuclear protein that participates in flowering time control by regulating the expression of FLC.The down-regulation of FLC was not due to PRC2activity;rather,it was likely the result of PRC1 activity,which is associated with AtBMI1C.

Materials and Methods

Plant materials and growth conditions

Arabidopsis thaliana plants(ecotype Columbia[Col–0])were used in this study.Seeds were surface-sterilized with2.25%NaHClO and plated on1X Murashige and Skoog(MS)basal salt medium containing0.3%agar and1%(weight/volume)sucrose.After stratification in the dark at4u C for two days,the plates were transferred to a growth chamber(Percival Scientific)set to 150m mol m22s21.The plants used for the flowering time determination were grown in a greenhouse at22u C(100m mol m22s21cool white fluorescent light)or18u C(dark).The seedlings sampled for the assay were grown under different conditions as indicated.

Flowering time determination

For flowering time measurement,mutant and wild-type(Col–0) plants were grown under long-day(LD;16h of light/8h of darkness at22u C)or short-day(SD;8h of light/16h of darkness at18u C)conditions.

Isolation of the AtBMI1C T-DNA insertion mutant

T-DNA insertion mutants of ATBMI1C(At3G23060)were ordered from the Arabidopsis Biological Resource Center(ABRC). Primers specific for sequences upstream and downstream of the T-DNA insertion were designed that could amplify the gene fragment without the T-DNA insertion.To amplify the T-DNA insertion,the T-DNA-specific primer LBb1(59-GCGTGGAC-CGCTTGCTGCAACT-39)and a gene-specific primer were used. We used two combinations of primers,each consisting of gene-specific primers,and a combination of a gene-specific primer and T-DNA-specific primer to identify individuals that were homozy-gous or heterozygous for the T-DNA insertion.The position of the T-DNA insertion was determined by sequencing those products carrying T-DNA-genome junctions.

amiRNA interference(amiRNAi)

creating amiRNAi constructs according to a previously published protocol[38].The resulting three constructs were delivered to wild-type plants via Agrobacterium-mediated transformation to generate AtBMI1C amiRNAi lines(AtBMI1C-Rs).

Vector construction and transformation

The coding sequence of AtBMI1C was amplified from the cDNA of wild-type(Col-0)plants using the primer pair and sequenced.Next,the fragment was cloned into the binary vector pCambia1300harboring the CAULIFLOWER MOSAIC VIRUS(CaMV)35S constitutive promoter and Yellow Fluores-cent Protein(YFP)gene using Xba I and Bam HI to produce the construct p35S::AtBMI1C-YFP.P35S::AtBMI1C without the YFP tag was also constructed to assess the effects of YFP on the function of AtBMI1C.Constructs for the expression of GFP-tagged AtBMI1C driven by the AP1,SUC2,and KNAT1

Figure8.ChIP analysis of H3K4me3and H3K27me3at FLC chromatin.(A)H3K4trimethylation levels at FLC chromatin between wild type and an ATBMI1C-overexpressing line harboring p35S::AtBMI1C-YFP.(B)H3K27trimethylation levels at FLC chromatin between wild type and AtBMI1C-overexpressing lines harboring p35S::AtBMI1C-YFP.Anti-H3K4me3or-H3K27me3antibodies were used for the assay.The locations of the primer pairs used to amplify FLC fragments across the region are indicated in(C).The values are the mean6SD of three independent experiments.

doi:10.1371/journal.pone.0021364.g008

The lengths of the promoters were chosen according to the indicated reference.

To create stable transgenic materials,wild-type(Col-0)plants were transformed using Agrobacterium tumefaciens GV3101[39]. Independent transgenic lines were obtained on selective MS medium containing hygromycin.T2or T3lines were used for flowering time determination and other assays. Semiquantitative RT-PCR and quantitative real-time RT-PCR

About100mg of ten-day-old seedlings grown under LD conditions were ground in liquid nitrogen.Total RNA was extracted from the seedlings using Takara RNAiso Plus(D9108A) according to the manufacturer’s protocol.The RNA was treated

Three micrograms of total RNA were used for the synthesis of full-length first strand cDNA with a RevertAid First Strand cDNA Synthesis Kit(Fermentas,K1622)according to the manufacturer’s protocol.One microliter of cDNA was utilized for semiquantita-tive RT-PCR.

Quantitative real-time RT-PCR was performed using Takara SYBR Premix Ex Taq in a7500fast real-time PCR instrument (Applied Biosystems).The assays were done according to the manufacturer’s instructions.ACTIN2/7was used as an endoge-nous control.

Observation of YFP fluorescence by confocal microscopy P35S::ATBMI1C-YFP transgenic lines(T3)were generated for the subcellular analysis of AtBMI1C.A Zeiss Meta confocal microscope was used to detect YFP fluorescence in the roots of ten-day-old seedlings and petals.Images in the YFP,DAPI,and brightfield channels were acquired.The final images were visualized using LSM510software.

Yeast two-hybrid analysis

cDNA from AtBMI1C and AtRING1A or B were cloned into pGADT7or pGBKT7and co-transformed into yeast strain AH109. Transformation,yeast growth,and quantitative b-galactosidase assays were conducted according to the protocols in the Clontech Yeast Protocols Handbook.

Protein pull-down assay

AtBMI1C was amplified and cloned into the bacterial expression vector pMAL-c2X(NEB)using Bam HI and Sal I.The constructs were transformed into BL21plus competent cells for protein expression.The proteins were induced overnight at16u C. MBP and MBP-AtBMI1C were purified using amylase resin (NEB).Plant total protein was extracted using extraction buffer (50mM Tris-HCl[pH7.6],150mM NaCl,2mM EDTA [pH8.0],1mM PMSF,and complete protease inhibitor cocktail) then mixed with protein-coated beads and incubated for2h at 4u C.The beads were then washed three times with extraction buffer.The bound proteins were eluted with SDS-PAGE sample loading buffer.The eluted proteins were separated by SDS-PAGE for Western blotting.Anti-Myc(Sigma)and-MBP(NEB) antibodies were used in this assay.

Chromatin immunoprecipitation(ChIP)analysis Constructs carrying p35S::ATBMI1C-YFP were transformed into wild-type plants,and homozygous lines were identified at the T3 generation for ChIP analysis.ChIP was performed as described [40]using ten-day-old seedlings grown on MS medium under LD conditions.Anti-H3K27me3and-H3K4me3antibodies were purchased from Upstate Biotechnology.Quantitative real-time PCR was performed to detect the FLC regions harboring H3K27me3and H3K4me3modifications with the primer pairs shown in Table5.The locations of the primer pairs are given in Figure8C.All ChIP assays were performed three times using at least three biological replicates.

Western blotting

Total protein was extracted using protein extraction buffer (50mM Tris-HCl[pH7.5],150mM NaCl,10mM MgCl2,and 1%NP-40)containing complete protease inhibitor cocktail (Roche),separated by SDS-PAGE,and probed with anti-GFP and-tubulin antibodies,respectively.

Table5.Primers used in this study.

Primer name Primer sequence(59to39)

ATBMI1C-SpeI-F ACTAGTTGGGAATCGGAGAGAAAGATGTTA ATBMI1C-BamHI-R GGATCCCTTCAGAGGCAGAGCCAGAGTCAGAG ATBMI1C-p1969-F CTGCAGCGTACGACCATTCAATTCTTGC ATBMI1C-p969-R TCTAGACTTTCTCTCCGATTCCCAAAC

SUC2-F AAAATCTGGTTTCATATTAATTTCA

SUC2-R ATTTGACAAACCAAGAAAGTAAGA

KNAT1-F GATCTAGAGCCCTAGGATTTGA

KNAT1-R ACCCAGATGAGTAAAGATTTGAG

AtBMI1C-RTF CATGCCTTGCTTGTCCAATC

AtBMI1C-RTR GCTTCGTCCAATCCATTGTC

Actin2/7F AGGCACCTCTTAACCCTAAAGC

Actin2/7R GGACAACGGAATCTCTCAGC

Actin2/7rtF GGTGTCATGGTTGGTATGGGTC

Actin2/7rtR CCTCTGTGAGTAGAACTGGGTGC

FLC-F CCTCTCCGTGACTAGAGCCAAG

FLC-R AGGTGACATCTCCATCTCAGCTTC

FT-F ACTATAGGCATCATCACCGTTCGTTACTCG

FT-R ACAACTGGAACAACCTTTGGCAATG

FLC P1F GCATTAGGTTGTTCCCTCCAAAC

FLC P1R GCCCTACCCATGACTAACGTGAG

FLC P2F GTTCGGGAGATTAACACAAATAATAAAGG FLC P2R GAAAACAAGCTGATACAAGCATTTCAC

FLC P3F TGGGGGTAAACGAGAGTGATG

FLC P3R GCAATAGTTCAATCCGTATCGTAGG

FLC B1F TGTTCTCAATTCGCTTGATTTCTAGT

FLC B1R GCCCGACGAAGAAAAAGTAGATAG

FLC B2F CGAGCACGCATCAGATCG

FLC B2R GGCGGATCTCTTGTTGTTTCTC

FLC B3F GACGTGCATATACAAATCCAAGAGAAC

FLC B3R CTTTGAATCACAATCGTCGTGTG

FLC B4F CCTCTCCGTGACTAGAGCCAAG

FLC B4R CTTCAACATGAGTTCGGTCTGC

FLC B5F CCTTGGATAGAAGACAAAAAGAGAAAGTG FLC B5R AGGTGACATCTCCATCTCAGCTTC

doi:10.1371/journal.pone.0021364.t005

using extraction buffers I(0.4M sucrose,10mM Tris-HCl [pH8.0],10mM MgCl2,5mM b-ME,0.1mM PMSF,and complete protease inhibitor cocktail),II(0.25M sucrose,10mM Tris-HCl[pH8.0],1%Triton X-100,10mM MgCl2,5mM b-ME,0.1mM PMSF,and complete protease inhibitor cocktail), and III(1.7M sucrose,10mM Tris-HCl[pH8.0],0.15%Triton X-100,2mM MgCl2,5mM b-ME,0.1mM PMSF,and complete protease inhibitor cocktail)in turn.The chromatin was treated overnight with0.4M H2SO4at4u C and the proteins were precipitated with25%trichloroacetic acid.The precipitate was washed three times with acetone,air-dried,and resuspended in 4M urea.The histone-enriched protein extracts were separated by15%SDS-PAGE,transferred to a PVDF membrane,and probed with anti-ubiquitin and-H3antibodies,respectively.Alignment and phylogenetic analysis

A phylogenetic tree of RING domain-containing proteins and RING domains from different organisms was constructed using MEGA4software[42].The RING domains of the RING domain-containing proteins were aligned using Jalview through ClustalW [43].

Acknowledgments

We thank Dr.Jessica Habashi for critical reading of the manuscript,and the ABRC for the T-DNA insertion lines.

Author Contributions

Conceived and designed the experiments:LM ZW XW SC.Performed the experiments:WL ZW JL HY XW.Analyzed the data:WL ZW JL HY XW SC LM.Contributed reagents/materials/analysis tools:LM SC.Wrote the paper:XW LM.

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