At-MINI ZINC FINGER2 and Sl-INHIBITOR OF MERISTEMACTIVITY, a ConservedMissing Link in the Regulation of FloralMeristem Termination in Arabidopsis and Tomato

Norbert Bollier,a Adrien Sicard,b Julie Leblond,a David Latrasse,c Nathalie Gonzalez,a Frédéric Gévaudant,a

Moussa Benhamed,c Cécile Raynaud,c Michael Lenhard,b Christian Chevalier,a Michel Hernould,a,1

and Frédéric Delmasa

a UMR1332 BFP, INRA, Université de Bordeaux, 33882 Villenave d’Ornon Cedex, Franceb Institut für Biochemie und Biologie, Universität Potsdam, 14476 Potsdam-Golm, Germanyc Institut of Plant Sciences Paris-Saclay (IPS2), UMR 9213/UMR1403, CNRS, INRA, Université Paris-Sud, Université d’Evry, UniversitéParis-Diderot, Sorbonne Paris-Cité, 91405 Orsay, France

ORCID IDs: 0000-0002-3946-1758 (N.G.); 0000-0002-5231-8120 (C.R.); 0000-0002-5727-6206 (C.C.); 0000-0002-2722-7032 (M.H.);0000-0002-2599-6778 (F.D.)

In angiosperms, the gynoecium is the last structure to develop within the flower due to the determinate fate of floral meristem(FM) stem cells. The maintenance of stem cell activity before its arrest at the stage called FM termination affects the numberof carpels that develop. The necessary inhibition at this stage of WUSCHEL (WUS), which is responsible for stem cellmaintenance, involves a two-step mechanism. Direct repression mediated by the MADS domain transcription factorAGAMOUS (AG), followed by indirect repression requiring the C2H2 zinc-finger protein KNUCKLES (KNU), allow for thecomplete termination of floral stem cell activity. Here, we show that Arabidopsis thalianaMINI ZINC FINGER2 (AtMIF2) and itshomolog in tomato (Solanum lycopersicum), INHIBITOR OF MERISTEM ACTIVITY (SlIMA), participate in the FM terminationprocess by functioning as adaptor proteins. AtMIF2 and SlIMA recruit AtKNU and SlKNU, respectively, to form a transcriptionalrepressor complex together with TOPLESS and HISTONE DEACETYLASE19. AtMIF2 and SlIMA bind to the WUS and SlWUSloci in the respective plants, leading to their repression. These results provide important insights into the molecularmechanisms governing (FM) termination and highlight the essential role of AtMIF2/SlIMA during this developmental step,which determines carpel number and therefore fruit size.

INTRODUCTION

The fruit, a specialized organ providing a suitable environment forseed maturation and dispersion, results from the development ofthe ovary following successful flower pollination and fertilization(Seymour et al., 2013). The ovary, along with the style and stigma,forms single or compound pistils, which constitute the gynoecium.The number of carpels arising from the floral meristem (FM), andconsequently the number of fruit locules, is determined during FMtermination, a stage at which stem cell activity is arrested (Lenhardet al., 2001).

In Arabidopsis thaliana, the transcription factor WUSCHEL(WUS) specifies the maintenance of stem cell activity in the shootapical meristem and FM (Mayer et al., 1998). In cooperation withtheFM regulator LEAFY,WUSactivates theAGAMOUS (AG) genein stamen and carpel primordia (Lenhard et al., 2001; Lohmannet al., 2001). AG then initiates reproductive development and atthe same time antagonizes WUS activity to terminate meristemactivity (Lenhard et al., 2001). The complete repression of WUSexpression is then essential to promote sharp developmental

transitions and regulate the total number of flower organs (Sunet al., 2009). Indeed, the loss of function of WUS results in theproduction of flowers lacking carpels and most stamens in Ara-bidopsis, while a gain of function of WUS leads to an increasednumber in floral organs and in some cases, the reiteration ofa flower inside the flower (Xu et al., 2005).The MADS box transcription factor AG thus plays a central

role in the repression of WUS during flower development, ac-cording to a two-step process. First, AG directly represses theWUS locus during the early stages of FM termination (stage 3)(Liu et al., 2011). The binding of AG to the WUS locus mediatesthe recruitment of CURLY LEAF, a core component of thePolycomb Repressive Complex 2 (PRC2), and the subsequentdeposition of Histone H3-lysine27 trimethylation (H3K27me3)repressive marks. PRC1 components, namely, TERMINALFLOWER2/LIKE HETEROCHROMATIN PROTEIN1, then rec-ognize the H3K27me3 marks and promote the compaction ofchromatin to ensure the stable repression ofWUS. However, thismechanism is insufficient for FM termination. Indeed, the com-plete arrest of WUS expression at floral stage 6 requires anadditional transcription factor belonging to the C2H2 zinc-fingerprotein family, named KNUCKLES (AtKNU) (Payne et al., 2004;Sun et al., 2009). The expression of AtKNU is directly induced byAG at stages 5 and 6, and a mutation in AtKNU results in anincreased number of carpels and stamens due to the prolongedexpression of WUS (Sun et al., 2009). Other factors are also

1Address correspondence to michel.hernould@inra.fr.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Michel Hernould (michel.hernould@inra.fr).www.plantcell.org/cgi/doi/10.1105/tpc.17.00653

The Plant Cell, Vol. 30: 83–100, January 2018, www.plantcell.org ã 2018 ASPB.

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required to fine-tune floral stem cell activities (reviewed in Sun andIto, 2015). However, the mechanism by which AtKNU repressesWUS remains unknown, and whether the above mechanisms canbe extended to other plant species needs to be investigated.

In tomato (Solanum lycopersicum), nucleotidic polymorphismsin a 15-bp repressor element localized downstream of the SlWUSlocus and sharing similarity with the CArG element of Arabidopsisunderlie variations in fruit locule number betweencultivars (Muñoset al., 2011; van der Knaap et al., 2014; Rodríguez-Leal et al.,2017). The increase in fruit locule number associated with thispolymorphism results in an approximate 30% yield increase interms of fruit production. Despite its inferred agronomical interest,themechanism underlying SlWUS regulation in tomato, andmoregenerally FM termination, is still not well understood. We pre-viously demonstrated that the INHIBITOR OF MERISTEM AC-TIVITY (SlIMA) protein, which belongs to the MINI ZINC FINGER(MIF) protein family, is involved in FM termination in tomato byfunctioning as an indirect repressor of SlWUS (Sicard et al., 2008).To date, the role of AtMIF2, the Arabidopsis homolog of SlIMA, inthe FM termination process is unclear.

Despite having a similar organization corresponding to a pistilresulting from the fusion of two carpels, the gynoecium in flowersof Arabidopsis and the wild tomato ancestor (Solanum lyco-persicum cv cerasiforme) matures after fertilization into a drysilique and a fleshy berry made of two seed-bearing locules, re-spectively. Here, to investigate the conservation and/or di-vergence in themolecularmechanisms regulatingFMtermination,we used a back-and-forth approach between Arabidopsis andtomato. We demonstrate that AtMIF2 and its tomato ortholog,SlIMA, are activated during flower development by AGand TomatoAGAMOUS1 (TAG1), respectively. AtMIF2 and SlIMA interact withthe transcriptional repressors AtKNU and SlKNUCKLES (SlKNU),respectively, allowing for the recruitmentof a chromatin remodelingcomplex at theWUS locusand its subsequent repression. In light ofthese observations and regarding the specific structure of MIFproteins (Hu et al., 2008), we propose that AtMIF2 and SlIMA act asadaptor proteins between transcriptional regulators and chromatinremodeling proteins to ensure the proper termination of stem cellsactivity within FM in angiosperm species.

RESULTS

AtMIF2 Is the Arabidopsis Ortholog of SlIMA

Since SlIMA is a MIF protein involved in the regulation ofFM termination in tomato (Sicard et al., 2008), we investigatedthe putative conservation of this function in Arabidopsis. TheMIF protein family in Arabidopsis encompasses three members:AtMIF1, AtMIF2, and AtMIF3 (Hu and Ma, 2006). AtMIF1 andAtMIF3 are mostly expressed in vegetative parts of the plant,whereasAtMIF2 ispreferentially expressed in reproductiveorgans(Arabidopsis eFP browser; Hu and Ma, 2006; Winter et al., 2007).Weassessed theexpression levelsofAtMIF1,AtMIF2, andAtMIF3in Arabidopsis during floral development using qRT-PCR (Figure1A). AtMIF2 expression increased gradually during flower de-velopment, while AtMIF1 and AtMIF3 expression was barely de-tectable at all developmental stages examined. This expression

pattern suggested that AtMIF2, like SlIMA in tomato (Sicard et al.,2008), is also involved in the regulation of flower development inArabidopsis.AtMIF2 transcripts were detected very early during floral de-

velopment, from stage 2 until stage 5, in the apical part of the FMand at layers 1 to 3 (Figure 1B). At stage 6, corresponding to theinitiation of carpel primordia, the expression signal became moreintense within the central cells of the meristem between the twocarpel primordia (Figure 1B). Once the formation of the fourwhorlsis initiated, i.e., from stage 6 until the end of floral development,AtMIF2 expression was restricted to the developing ovules in theovary (Figure1C).AtMIF2expression thusappears tobe regulatedaccording to a precise pattern during floral development and ismainly associated with early carpel and ovule development.To examine whether AtMIF2 is involved in the regulation of

flower development, we studied the effects of gene silencing.T-DNA insertionmutant lines in AtMIF2 have not yet been found inArabidopsis, nor have constitutive knockdown approaches (RNAior antisense lines) succeeded in producing transformed plantswith a strong reduction in AtMIF2 transcript level. Since AtMIF2 isstrongly expressedduring ovule/seeddevelopment, we reasonedthat a complete loss of function would likely impair the devel-opment of transgenic seedlings. Therefore, we decided to reduceAtMIF2 expression specifically during flower development but notduring ovule/seed development. To this end, we expressed anartificial microRNA (Schwab et al., 2006; Ossowski et al., 2008)specifically directed against AtMIF2 mRNA under the control ofan 800-bp-long sequence from the PISTILLATA promoter (ProPI)(Honma and Goto, 2000). The ProPI:amiRNA-AtMIF2 transgeniclines were thus generated. As a prerequisite, we confirmed thatProPIdrives strongGUS reporter gene expression in young flowerbuds and within the inflorescence meristem, notably at thestage when FM termination occurs, whereas no expression wasdetected during ovule/seed development (Supplemental Figures1A to 1D).In ProPI:amiRNA-AtMIF2 plants, AtMIF2 expression was

strongly reduced comparedwithwild-typeCol-0*Lerplants (;10-fold before stage 9;;19-fold during stages 10–13;;4-fold duringstages 13–16) (Figure 2A). Flowers from ProPI:amiRNA-AtMIF2plants displayed an increased number of locules compared withthe two locules of thewild type. As a consequence, 30 to 58%and11 to 20% of developed ProPI:amiRNA-AtMIF2 siliques weretrilocular and tetralocular, respectively, containing three and fourrows of seeds separated by three or four septa (Figures 2B to 2G).Such an indeterminate phenotype inducing multicarpellar fruitwas also obtained in Pro35S:SlIMA-RNAi tomato plants (Figure2I). InPro35S:SlIMA-RNAiplants, around20%of fruitsweremadeof three to eight carpels instead of two in thewild type (Figures 2Hand 2I) due to the strong reduction in SlIMA transcript abundance(;5-fold compared with the wild type) (Supplemental Figure 1E).Taken together, these results indicate that AtMIF2 in Arabidopsisand SlIMA in tomato are involved in determining carpel number.

Isolation and Characterization of the KNU Orthologin Tomato

An increase in carpel number was previously described in severalArabidopsis mutants that belong to theWUS repression pathway

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duringFMtermination, suchas the knumutant (Payneet al., 2004).TheC2H2ZINC-FINGERproteinAtKNU regulates FM terminationinArabidopsis through the repressionofWUS (Liu et al., 2011).Wenext investigated whether the function of KNU is conserved be-tween Arabidopsis and tomato.

For this purpose, we first searched for putative homologs ofAtKNU in the available full tomato genome sequence (TomatoGenome Consortium, 2012). Three distinct homologous se-quences were identified: one named Solyc00g014800 at positionSL2.50ch00:11060181-11060672, localized on the artificialpseudomolecule composed of scaffolds that could not be placed

on either one of the 12 tomato chromosomes defined as chro-mosome 0 (Tomato Genome Consortium, 2012); one on chro-mosome 12 at positions SL2.50ch12:51560501-51562100(annotated C2H2_SL2.50Ch12); and one on chromosome 2 atpositionsSL2.50ch02:54952316-54952822(annotatedSl-KNUCKLES)(Supplemental Figures 2A and 2B). These three sequences dis-played a lowbut significant level of identity with AtKNU (27, 27, and33%, respectively), highlighting the presence of a conservedC2H2zinc-finger domain (Supplemental Figure 2B). However, the twoformersequencesdidnotexhibitaconservedEARdomain,which isone of the characteristics of AtKNU protein (Payne et al., 2004)(Supplemental Figure 2B).Moreover, our own functional analysis ofSolyc00g014800, either by overexpression or RNAi silencing, didnot lead to any phenotypical alteration in flower development in

Figure 2. Fruit Phenotypes of AtMIF2 and SlIMA Loss-of-Function Plantsin Arabidopsis and Tomato, Respectively.

(A) Expression analysis of AtMIF2 in three independent ProPI:amiRNA-AtMIF2 plants comparedwith Col*Lerwild-type plants at various stages offlower development using qRT-PCR. Error bars represent SD of three bi-ological replicates, and asterisks indicate significant differences from thecontrol (Col*Ler) using two-tailed t test (*P

tomato. Only the third sequence localized on chromosome 2 fit theminimal requirements to be considered a potential AtKNU homo-log, i.e., the presence of a C2H2 zinc finger and an EAR domain(Supplemental Figure 2B). In addition, this gene was only expressedduring floral and fruit development and more particularly in carpels(Figures3Aand3B). InyoungFM,thisgenewasstronglyexpressedinthe first layers of sepal and petal primordia at stage 2, in all primordiaat stage 6, and in ovules and anthers at stage 9 (Figure 3A). Sincethis expression pattern is consistent with the expression of AtKNUin Arabidopsis, this sequence was then tentatively named SlKNU.

To investigate the function of SlKNU, we generated transgenictomato with up- or downregulated expression of this gene(Supplemental Figure 2C). The overexpression of SlKNU usinga constitutive promoter (Pro35S:SlKNU) was correlated withseveral phenotypic alterations suchasa lossof apical dominance,resulting inadwarf andbushyphenotypeanda reduction inoverallflower size (Figures 3C and 3D). Conversely, the Pro35S:SlKNU-RNAi silencing lines did not display any vegetative phenotype butwere strongly affected in flower development (Figure 3D, rightpanel). The number of petals, stamens, and carpels was higher inflowers of the Pro35S:SlKNU-RNAi lines compared with the wildtype (Figure 3D). These lines producedmulticarpellar fruits (Figure3E), with up to eight carpels in several fruits in the most extremeline (Figure 3F). Altogether, these results indicate that SlKNU is thelikely ortholog of AtKNU.

AtMIF2 and SlIMA Are Members of the Genetic ProgramGoverned by AG/TAG1

Wenext investigatedthe transcriptional regulationofAtMIF2and/orSlIMA to test their involvement within the genetic frameworkcontrolling FM termination. First, we usedmVISTA software (http://genome.lbl.gov/vista/index.shtml) to perform the alignment ofa ;2.8-kb-long DNA sequence encompassing the 59 upstreamgene and coding sequence of AtMIF2with the corresponding DNAsequence of the SlIMA locus in order to evaluate the phylogeneticconservationbetween the twosequences.Within the2385-bp-longsequence upstream of the translation start codon of AtMIF2 andSlIMA, five conserved DNA regions referred to as domain A to Ewere identified (Supplemental Figure 3A). We then searched forpotential transcription factor binding sites using rVista and Mat-Inspector software (https://www.genomatix.de/matinspector).Based on the phenotypic alterations induced by the silencing ofAtMIF2 and SlIMA, we focused on the identification of bindingsites for transcription factors involved in carpel identity and FMdetermination. Putative binding sites for AGandAGAMOUS-LIKE(AGL) proteins were found in the promoter sequences of bothAtMIF2 and SlIMA (Supplemental Figure 3B). Among these, AG,AGL1, and AGL3 binding sites, located at around 230 nucleotidesupstream of the translation start codon, overlapped with the con-served B domain. A second AG binding site was identified at closeproximity to the conserved E domain in both promoter sequences(Supplemental Figure 3B). The sequences of these putative AGbinding sites identified in the conserved E (TTCCAAATTAGATA)and B (AACCCTAGATGTC) domains are noncanonical CArG-boxes (Huangetal., 1993)andcouldbeweakbindingsites,but theirpresence, togetherwith the known functionofAGandAGLproteinsin FM termination and carpel development, suggested that these

proteins could regulate SlIMA/AtMIF2 expression during flowerdevelopment.To determine whether AG interacts with these sequences in the

AtMIF2 promoter, we tested the relevance of these putative AGbinding site using electrophoretic mobility shift assay (EMSA)(Figure 4A). First, soluble AG proteins were mixed with an oligo-nucleotide sequence (Supplemental Data Set 1) used as a positivecontrol probe for AG fixation as previously reported (Riechmannet al., 1996). A mobility shift was observed which could not beobtained when the control probe was mixed in the presence ofsolubleproteins fromcells thatdidnotexpressAG(usedasnegativecontrol protein cell extracts) (Figure 4A, left panel). We then testedtwo oligonucleotide sequences, designated as pAtMIF2-E andpAtMIF2-B (Supplemental DataSet 1), respectively, correspondingto theEandBconserveddomains identified asputativeAGbindingsites (Supplemental Figure 3B). In the presence of AG proteins,a mobility shift with either pAtMIF2-E or pAtMIF2-B probe wasobtained, similar to that observed with the positive control probe(Figure4A,center and rightpanels), thus indicating thatAGcanbindto the two pAtMIF2 oligonucleotide sequences examined.To evaluate the involvement of AG/TAG1 (Pnueli et al., 1994) in

the regulation of AtMIF2/SlIMA expression, we determined therelative abundance ofAtMIF2 and SlIMA transcripts in plants withmodified expression of AG and TAG1. The expression of AtMIF2was totally abolished in flower buds of the ag-3 Arabidopsismutant (Figures4Band4C),while thatofSlIMAwasupregulated intomato Pro35S:TAG1 plants overexpressing TAG1 (Figure 4D;Supplemental Figure 4). The results ofGUS reporter gene assayswere in full agreement with these results: When expressed in thePro35S:TAG1 background, GUS expression driven by the SlIMApromoter (ProSlIMA:GUS) was greatly enhanced in tomato fruits(Figure 4E).To analyze whether the conserved CArG-box sequence in the

SlIMA promoter has any relevance to its expression during flowerand fruit development in tomato, we designed two single-guideRNAs (sgRNAs) targeting the region of the putative CArG-box toinduce mutations using CRISPR/Cas9 technology. After trans-formation, we obtained three independent T0 transgenic linesdisplaying homozygous mutations within the SlIMA promoter, asrevealed by sequencing (Figure 4F). Two of these lines weremutated at the putative CArG-box site (CR#1-ProSlIMA andCR#2-ProSlIMA), and the third line was mutated further down-stream (CR#3-ProSlIMA). These three lines did not display anyvegetative defects. Interestingly, the CR#1-ProSlIMA and CR#2-ProSlIMA lines displayed an alteration in fruit development, withasignificantly increasednumberof loculescomparedwith thewildtype (Figures 4G and 4H). The third line, CR#3-ProSlIMA, did notdisplay any alteration in fruit development (Figures 4G and 4H).Using qRT-PCR, we found that SlIMA expression was repressedin CR#1-ProSlIMA and CR#2-ProSlIMA flowers by 5- and 2-fold,respectively, compared with wild-type plants, while SlIMA ex-pression was not significantly affected in CR#3-ProSlIMA (Figure4I), indicating that the CArG-box is necessary for SlIMA expres-sion in tomato flowers. Altogether, these results suggest that AGand TAG1 regulate AtMIF2 and SlIMA expression, respectively,either directly or indirectly through the activation of other MADSbox protein(s) that could act as positive regulators of AtMIF2 andSlIMA expression.

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The Misexpression of AtMIF2/SlIMA or AtKNU/SlKNUAffects the Expression Pattern of WUS and SlWUS inArabidopsis and Tomato

In mutants producing additional carpels, such as clavata, FMtermination is often impaired, which is related to the ectopic ex-pression of WUS (Schoof et al., 2000). We therefore investigated

the origin of the formation of additional carpels in ArabidopsisProPI:amiRNA-AtMIF2 and in tomato Pro35S:RNAi-SlIMA si-lencing lines. This phenotype could be linked to an impairmentin FM termination, likely due to ectopic WUS expression. Wetherefore analyzed the temporal and spatial expression of WUSand SlWUS (Reinhardt et al., 2003) in AtMIF2 and SlIMA silencingplants. In ProWUS:GUS plants (Gross-Hardt et al., 2002), which

Figure 3. SlKNU Expression Pattern and Phenotypes of Pro35S:SlKNU and Pro35S:SlKNU-RNAi Tomato Plants.

(A) Expression analysis of SlKNU in developing tomato flower buds analyzed by in situ hybridization. se, sepal; pe, petal; st, stamen; ca, carpel; ov, ovule.Bars = 100 mm (S2 and S4) and 250 mm (S6 and S9).(B)Expressionanalysis ofSlKNUusingqRT-PCR indifferent tissues andduring floral and fruit development. daa, daysafter anthesis. Error bars represent SDof three biological replicates.(C) Vegetative phenotypes of wild-type and two Pro35S:SlKNU lines. Bars = 5 cm.(D) Flowers of wild-type, Pro35S:SlKNU, and Pro35S:SlKNU-RNAi lines. Bars = 5 mm.(E) Fruits of wild-type, Pro35S:SlKNU, and Pro35S:SlKNU-RNAi lines. Bars = 5 mm.(F) Number of locules per fruit in wild-type and Pro35S:SlKNU-RNAi lines. Data are presented as percentage of fruits per locule number category (n =25 fruits). *P < 0.05 and **P < 0.01 (Tukey HSD).

AtMIF2/SlIMA Control WUS Expression 87

were used to report the endogenous expression pattern ofWUS,WUSwasexpressed inflowerbudsatstage5within theorganizingcenter of FM harboring the stem cells; at stage 6, its expressiontotally disappeared in this region (Figure 5A), as previously de-scribed (Mayer et al., 1998). WUS expression was expected tostart again in later floral developmental stages during anther andovule development (Gross-Hardt et al., 2002), which could explainthe detection ofWUS transcripts in these stages using qRT-PCR(Figure 5B). In the ProPI:amiRNA-AtMIF2 lines, WUS was ex-pressed at a level 2-fold higher in early flower buds at stages 1 to6 and;5-fold higher at later stages 6 to 10 comparedwith thewildtype (Figure 5B). These results are fully consistent with the per-sistence of a strong GUS signal in flower buds in pWUS:GUSreporter lines at stage 6 (Figure 5A). Similar data were obtained intomato, asSlWUSexpressionwasstrongly induced inflowerbudsofPro35S:SlIMA-RNAiplants comparedwith thewild type (Figure5C;Sicardetal., 2008)butalso in theCRISPRCR#1-ProSlIMAandCR#2-ProSlIMA lines (Figure 5D).Next, we examined whether SlKNU may play a similar role to

AtKNU in the repression ofWUS by analyzing SlWUS expressionin SlKNUmisexpressing tomato plants. In Pro35S:SlKNU plants,the expression of SlWUS was reduced compared with wild-typeplants at all stages of flower development examined (Figure 6A).Conversely, the expression of SlWUS was enhanced comparedwith the wild type in Pro35S:SlKNU-RNAi plants (Figure 6A). Inaddition, the expression of SlKNU was greatly enhanced asa result of TAG1 overexpression (Figure 6B), indicating that TAG1positively regulates SlKNU.Therefore, these data show that both SlKNU/AtKNU and

SlIMA/AtMIF2 are required to repress WUS expression duringFM termination.

AtMIF2 and SlIMA Interact Physically with AtKNU and SlKNU

Proteins from the MIF family are characterized by the presence ofa unique domain, a noncanonical zinc finger involved in protein-protein interaction (Hu and Ma, 2006; Hong et al., 2011), sug-gesting that these proteins may act through interaction withtranscription factors. Therefore, we assayed the ability of AtMIF2and SlIMA to interact with AtKNU and SlKNU using bimolecularfluorescence complementation (BiFC) experiments. As revealedby the fluorescent signal resulting from the reconstruction ofa functional YFP, AtMIF2 and SlIMA were able to interact with

Figure 4. AtMIF2/SlIMA Expression Is Activated by AG/TAG1.

(A) In vitro binding of AG to theAtMIF2promoter sequences analyzedbyEMSA. The black arrow points to themobility shift corresponding to AGbinding to the probe; the white arrow indicates an unspecific binding ofbacterial proteins to the probe. Control probe is a conserved CArGsequence as described by Riechmann et al. (1996). pAtMIF2-E andpAtMIF2-B: sequences from the AtMIF2 promoter (SupplementalFigure 3).(B) Expression analysis of AtMIF2 in wild-type Ler and ag-3 floral buds atvarious stages of floral development using qRT-PCR.(C) In situ hybridization against AtMIF2 mRNA in floral bud of ag-3 plant.Bars = 100 mm.(D) Expression analysis of SlIMA in wild-type and Pro35S:TAG1 tomatofloral buds at various stages of development using qRT-PCR.(E) GUS staining in fruits of ProSlIMA:GUS and ProSlIMA:GUS Pro35S:TAG1 double-transgenic lines. Bars = 5 mm.

(F) CRISPR/Cas9-induced deletions in the CArG motif (CArG-box) of theSlIMA promoter in three independent T0 plants. The three CR-ProSlIMAplants were homozygous for the deletion. Red font highlights sgRNAtargets, and bold indicates protospacer-adjacent motif (PAM) sequences.(G)Cross sections of fruit fromwild-type,CR#1-ProSlIMA,CR#2-ProSlIMA,and CR#3-ProSlIMA lines.(H) Number of locules per fruit produced by wild-type and CR#ProSlIMAlines. Data are presented as percentage of fruits per locule number cat-egory (n = 12 fruits). **P < 0.01 (Tukey HSD).(I)Expression analysis of SlIMA in wild-type andCR#-ProSlIMA floral budsusing qRT-PCR.(B), (D), (H), and (I)Error bars represent SD of three biological replicates andasterisks indicate significant differences from the control (WT) using two-tailed t test (*P < 0.05, **P < 0.01, and ***P < 0.001).

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AtKNU and SlKNU (Figures 7A and 7B). The interacting proteinswere exclusively localized to the nucleus in this heterologoussystem, like AtMIF2/SlIMA and AtKNU/SlKNU, which accumu-lated inside the nucleus in homologous systems (SupplementalFigure 5). We made use of truncated forms of KNU and SlKNU toinvestigate which part is involved in the KNU-MIF interaction.When theC2H2zinc-fingerdomainand its immediate surroundingregion were removed, a nucleocytoplasmic localization of theprotein was found, whereas when this region was present, thenuclear localization of the protein was observed (SupplementalFigure 6A). Interestingly, SlIMA and AtMIF2 interacted with thetruncated proteins SlKNUDC43-168 and AtKNUDC39-161, re-spectively (Supplemental Figure 6B), but not with any of theN-terminal truncated forms, thussuggesting thatSlIMAandAtMIF2interact specifically with the N-terminal part of SlKNU and AtKNU,respectively.We also investigated this interaction using an in vitro pull-down

assay. The purified recombinant bait protein 6xHis-AtKNU wasattached toaNi-NTAcolumn to trap theGST-AtMIF2preyprotein.The GST-AtMIF2 protein was effectively pulled down in thepresence of 6xHis-AtKNU (Figure 7C). The interactions betweenAtMIF2 and AtKNU, as well as SlIMA and SlKNU, were alsoconfirmed in a heterologous system using a yeast two-hybridassay (Figure 7D). These interactions likely occurred via the C2H2zinc-finger domain within the N-terminal part of AtKNU andSlKNU, since when the EAR domain was deleted from theC-terminal parts of these proteins, the AtMIF2-AtKNU and SlIMA-SlKNU interactions were conserved (Figure 7E; SupplementalFigure 7). Taken together, these data demonstrate that AtMIF2/SlIMAdirectly interactwith AtKNU/SlKNUboth in vitro and in vivo.

AtMIF2/SlIMA Mediate AtKNU/SlKNU andTPL/SlTPL1 Interactions

The presence of an EAR domain at the C terminus of ArabidopsisKNU is an important characteristic in the mediation of tran-scriptional repression (Kagale and Rozwadowski, 2011). Thisrepressive activity of AtKNU is thought to require the interactionwith a transcriptional corepressor such as TOPLESS (TPL), whichcan indeed interact (although weakly) with AtKNU, as shown intargeted yeast two-hybrid experiments (Causier et al., 2012) butno fluorescence indicating in vivo interaction could be observedusing BiFC (Supplemental Figure 8). As proposed by Kagale andRozwadowski (2011), the presence of an additional protein couldbe required for the stabilization of the interaction. To investigatewhether AtMIF2 could play such a role as an adaptor protein, weinvestigated the interactionbetweenAtKNUandTPLandbetweenSlKNU and the tomato ortholog of the Arabidopsis TPL protein,

Figure 5. AtMIF2 and SlIMA Repress WUS and SlWUS Expressionduring FM Termination in Arabidopsis and Tomato, Respectively.

(A) and (B) Expression analysis of WUS in wild-type (Col*Ler)and AtMIF2 loss-of-function (ProPI:amiRNA-AtMIF2 and ProPI:amiRNA-AtMIF2 pWUS:GUS) plants in floral bud at various

developmental stages using ProWUS:GUS staining and qRT-PCR.Bars = 100 mm.(C) and (D) Expression analysis of SlWUS in the wild type, SlIMA silencingline (Pro35S:SlIMA-RNAi ), and CR#-ProSlIMA flower buds at stage 1-6using RT-PCR (C) and qRT-PCR (D).(B) to (D)Error bars represent SD of three biological replicates and asterisksindicate significant differences from the control (WT) using two-tailed t test(*P < 0.05, **P < 0.01, and ***P < 0.001).

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SlTPL1 (Hao et al., 2014), in the presence of AtMIF2 or SlIMA,respectively, by BiFC analysis. A BiFC YFP fluorescent signalindicating the formation of an AtKNU-TPL or SlKNU-SlTPL1complex was solely observed in the presence of AtMIF2 or SlIMA,here fused toRFPasa second reporter protein in the assay (Figure8A). The BiFC signal was localized in small subnuclear specklesexhibiting RFP signals, suggesting the colocalization of AtMIF2-RFPwith theAtKNU/TPL interacting proteins andSlIMA-RFPwithSlKNU/SlTPL1.

We then tested if AtMIF2/SlIMA indeed forms a tripartitecomplex using a yeast three-hybrid assay. When cotransformedwith the empty pYES2 plasmid (pYES), AD-TPL with BD-AtKNUand AD-SlTPL1 with BD-SlKNU were unable to induce yeastgrowth on selective medium (Figure 8B). However, the growth ofyeast colonies was observed in the presence of pYES2 plasmidsexpressing either AtMIF2 (pYES-AtMIF2) or SlIMA (pYES-SlIMA)(Figure 8B). These results suggest that AtMIF2 or SlIMA likelystabilize the AD-TPL/BD-AtKNU or AD-SlTPL1/BD-SlKNU complexes,allowing for the growth of yeast on selectivemedium. In addition,

the supplementation of 3-aminotriazole (3-AT; 75 mM), a com-petitive inhibitor of the HIS3 gene product, in the selectivemedium indicated that the tripartite complexes AD-TPL/BD-AtKNU/AtMIF2 and AD-SlTPL1/BD-SlKNU/SlIMA are stable.These results indicate that AtMIF2/SlIMA act as an adaptor/stabilizer to allow the bridging between TPL/SlTPL1 and thetranscription factor AtKNU/SlKNU.The mechanism by which TPL mediates gene repression was

recently studied, revealing its ability to interact with a HISTONEDEACETYLASE (HDA)-like HDA19 protein, suggesting that TPL-mediated regulation of gene expression involves a deacetylationmechanism (Szemenyei et al., 2008; Kagale and Rozwadowski,2011). BiFC experiments with Arabidopsis TPL and HDA19 pro-teins or their orthologs in tomato, SlTPL1 andSlHDA1 (Zhao et al.,2015), confirmed this interaction (Figure 8C). We also demon-strated that AtMIF2 and SlIMA were able to interact with HDA19and SlHDA1, respectively (Figure 8D), arguing for the hypothesisthat they function as adaptor proteins, thereby enabling the for-mation of a chromatin remodeling complex.

AtMIF2/SlIMA Bind to the WUS/SlWUS Locus in aKNU-Dependent Manner

To examine whether AtMIF2/SlIMA binds directly to the WUS/SlWUS locus, we performed chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) analyses using transgenicArabidopsis and tomato lines overexpressing AtMIF2-3HA andSlIMA-YFP, respectively. Commercial antibodies directedagainst the 3HA- or YFP-protein tag were used to immunopre-cipitate AtMIF2- or SlIMA-chromatin complexes from flower buds(a mixture of harvested buds from stages 1–12). Five and eightDNA sequences spread along theWUS locus (Liu et al., 2011) andthe SlWUS locus, respectively, were used to identify the chromatin-immunoprecipitated sequences (Figures 9A to 9C; primers listed inSupplemental Data Set 1). There was no apparent enrichment of thegenomic fragments in wild-type samples or with control anti-IgGantibodies (Figures 9B to 9D). In contrast, in Arabidopsis, among thefive targeted sequences, the P1 and P4 sequences were amplifiedafter chromatin enrichment from AtMIF2-HA expressing floral buds,demonstrating that AtMIF2 is able to bind to this sequencewithin theWUS locus (Figures 9B to 9D). In tomato, three of the eight targetedsequences (P1, P7, andP8)were amplified inSlIMA-YFP-expressingfloral buds (Figure 9C). Notably, these regions are at similar locationsin both species (Figure 9A).WhenAtMIF2-HAwasoverexpressed in the knumutant genetic

background, qPCR following chromatin immunoprecipitation didnot lead to any amplification for the different DNA sequencestargeted (Figure 9B), indicating the requirement of AtKNU ex-pression for AtMIF2 binding to the WUS locus.To confirm the binding of AtMIF2 and AtKNU to theWUS locus,

wealsomadeuseofDNAadeninemethyltransferase identification(DamID) technology (Germann et al., 2006), a technique used tomap the binding sites of DNA binding proteins based on theexpression of a protein fused with the DNA adenine methyl-transferase (Dam) and the detection of adenine methylation usinga methylation-specific qPCR protocol (Germann and Gaudin,2011). DNA was extracted from young inflorescences of Ara-bidopsis lines expressing AtMIF2 or AtKNU fused to the Dam

Figure 6. SlKNU Expression Is Promoted by TAG1 to Repress SlWUS inTomato Floral Bud.

(A) Expression analysis of SlWUS in Pro35S:SlKNU-RNAi and Pro35S:SlKNU plants, compared with wild-type plants, using qRT-PCR.(B) Expression analysis of SlKNU in three Pro35S:TAG1 overexpressinglines, compared with wild-type plants, using qRT-PCR.Error bars represent SD of three biological replicates and asterisks indicatesignificant differences from the control (WT) using two-tailed t test (*P <0.05 and **P < 0.01).

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(Dam-AtMIF2 and Dam-AtKNU, respectively) under an ethanol-inducible promoter or the Dam alone, prior to and after 24 h ofethanol induction (NI and I). Using the same five targeted se-quences as those used for ChIP-qPCR experiments as well asanother one (P3), qPCR was performed to determine the en-richment of methylation at the different sites indicating thebinding of the Dam-fusion protein. P1 and P4 sequences wereenriched in both Dam-AtMIF2 and Dam-AtKNU, indicating thebinding of AtMIF2-DmandAtKNU-Dam fusion proteins on theseregions of the WUS locus (Figure 9D). Together, these sug-gest that AtMIF2/SlIMA participate in the direct repression ofWUS/SlWUS.

To test thehypothesis that the repression ofWUSbyanAtKNU-AtMIF2-TPL-HDA complex requires histone deacetylation, weevaluated the effects of trichostatin A (TSA), a specific inhibitor ofhistone deacetylase activity, on WUS expression in ArabidopsisCol-0 wild-type plants and Pro35S:AtMIF2-overexpressingplants. TSA treatment led to a strong increase inWUS expressionin the wild type and (more importantly) partially rescued WUSexpression to wild-type levels in AtMIF2 overexpressing plants(Figure 9E), indicating that the repression of WUS requires

a deacetylation mechanism putatively involving AtMIF2. The highlevel of WUS expression in the hda19 mutant strongly supportsthis hypothesis (Figure 9E). To examine whether this increasedexpression level is associated with an extendedWUS expressionpattern,we treated theProWUS:GUSPro35S:AtMIF2 reporter linewith or without TSA beforeGUS staining. After TSA treatment, theGUS signal was observed in an enlarged zone of the shoot apicalmeristem compared with that obtained in TSA-untreated plants(Figure 9F), thus indicating a derepression ofWUS in conjunctionwith the inhibition of HDA activity.These findings indicate that the repression ofWUSmediatedby

AtMIF2 in Arabidopsis requires the activity of a histone deace-tylase such asHDA19, suggesting that AtMIF2 acts as an adaptorprotein to form a chromatin remodeling complex to epigeneticallyrepress WUS through histone deacetylation.

DISCUSSION

Floral stemcell termination involves a complex regulatory networkthat has been extensively described in the model Arabidopsis,but much less is known about the conservation of (or potential

Figure 7. Interaction Analyses between AtMIF2 and AtKNU and between SlIMA and SlKNU.

(A)BiFCanalysis testing the interactionbetweenAtMIF2andAtKNU (upperpanel) andSlIMAandSlKNU (lowerpanel) in onionepidermal cells.Bars=25mm.(B)BiFCanalysis testing the interactionbetweenAtMIF2andSlKNU (upperpanel) andSlIMAandAtKNU(lowerpanel) in onionepidermal cells. Bars=50mm.(C) In vitro pull-down of GST-AtMIF2 with 6xHis-AtKNU as a bait bound to Ni-NTA resin. GST-AtMIF2 was detected using anti-GST antibody. The blackarrow indicates GST-AtMIF2.(D)Yeast two-hybrid interactionswere testedby transforming fusionsofeitherAtMIF2orSlIMAwith theGal4activationdomain (AD) and fusionsofAtKNUorSlKNU to theGal4bindingdomain (BD).Serial dilutionsof yeast cells from105 to10onnonselectiveSDmedium lacking leucineand tryptophan (-L/-W)shownormal yeast growth. Only positive interactors are able to grow on restrictive growth medium supplemented with 25 mM 3-AT and lacking leucine,tryptophan, and histidine (-L/-W/-H).(E) BiFC analysis testing the interaction between AtMIF2 and AtKNU-EARdel (upper panel) and SlIMA and SlKNU-EARdel (lower panel) in onion epidermalcells. Bars = 50 mm.

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differences in) this mechanism in other plant species. Here,combining information from Arabidopsis and tomato, we char-acterized an actor in this process, AtMIF2/SlIMA, and showed itsconserved function in FM termination and carpel number control.

AtMIF2, the Floral Actor of the MIF Family in Arabidopsis, IsInvolved in Controlling Locule Number, Like SlIMAin Tomato

The three genes of the MIF family in Arabidopsis were originallydescribed as important actors in hormonal regulation. AtMIF1 andAtMIF3 are expressed in vegetative parts of plants (leaves androot), and AtMIF2 is expressed in stems and inflorescences (Huand Ma, 2006). Here, we precisely described the expressionpattern of AtMIF2, which is expressed during early stages of floraldevelopment. The lack of AtMIF2 knockout lines and the obser-vation that knockdownapproaches (RNAior antisense lines) failed

to produce transformed plants displaying a strong reduction inAtMIF2 transcript level has hampered the study of the biologicalfunction of AtMIF2, as acknowledged by Hu and Ma (2006). Theonly otherMIF gene studied so far is SlIMA in tomato (Sicard et al.,2008), for which partial loss-of-function plants could be obtainedusingbothRNAiandRNAantisensestrategies (Sicardetal., 2008),revealing the roleof thisgene infloraldevelopment.Here,using theProPI:amiRNA-AtMIF2 line, with reduced expression of AtMIF2during young floral bud development, we demonstrated thatAtMIF2, like SlIMA in tomato, functions early in carpel numberdetermination.

AtMIF2 and SlIMA Are Directly or Indirectly Regulated byAG/TAG1 to Repress WUS/SlWUS Expression

Since both the biological functions and expression patterns ofAtMIF2 in Arabidopsis and SlIMA in tomato are conserved, we

Figure 8. AtMIF2/SlIMA Bridges the KNU/SlKNU and TPL/SlTPL1 Interaction and Directly Interacts with HDA19/SlHDA1.

(A) BiFC analysis of the interactions between Arabidopsis KNU and TPL (upper panels) and tomato SlKNU and SlTPL1 (lower panels) in the presence ofAtMIF2 and SlIMA, respectively, in onion epidermal cells. Bars = 25 mm.(B)Yeast three-hybrid assay demonstrating the formationof AtMIF2-KNU-TPL andSlIMA-SlKNU-SlTPL1 trimeric complexes. Serial dilutions of yeast cellsfrom 105 to 10 on nonselective SD medium lacking leucine, tryptophan, and uracil (-L/-W/-U) show normal growth. Only positive interactions were able togrow on restrictive growth medium supplemented with 75 mM 3-AT and lacking leucine, tryptophan, and histidine (-L/-W/-H).(C) BiFC analysis of the interactions between TPL and HDA19 (left panel) and SlTPL1 and SlHDA1 (right panel) in onion epidermal cells. Bars = 50 mm.(D) BiFC analysis of the interactions between AtMIF2 and HDA19 (left panel) and SlIMA and SlHDA1 (right panel) in onion epidermal cells. Bars = 50 mm.

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Figure 9. AtMIF2/SlIMA Bind WUS/SlWUS Locus to Repress Their Expression via Histone Deacetylation.

(A) Schematic representation of theWUS and SlWUS loci (59 and 39UTR are in gray, introns in white, and exons in black) with the different targeted regions(P1 to P8).(B)ChIP assay at theWUS locus usingHA- or IgG-antibodies inCol-0, knu,Pro35S:AtMIF2-3HA inCol-0, andPro35S:AtMIF2-3HA in knuplants. The yaxisshows enrichment relative to input using IgG as a control. Error bars represent SD of three biological replicates, and asterisks indicate significant differencesfrom the control (Col-0) using two-tailed t test (*P < 0.05 and **P < 0.01).(C)ChIPassayat theSlWUS locususingGFPor IgGantibodies inwild-typeandPro35S:IMA-YFPplants. The yaxis shows relative enrichment to input usingIgGas acontrol. Error bars represent SD of three biological replicates, and asterisks indicate significant differences from the control (Col-0) using two-tailed ttest (*P < 0.05 and **P < 0.01).(D)DamID ratios at different regions of theWUS locus prior (NI) and after 24 h of ethanol induction (I) in lines expressing Arabidopsis MIF2 or KNU fused toDAM (Dam-AtMIF2 and Dam-KNU, respectively).(E) Expression analysis of WUS in Col-0, Pro35S:AtMIF2, and hda19 floral buds (stages 1–12) treated or not with 0.5 mM TSA, using qRT-PCR. Errorbars represent SD of three biological replicates and asterisks indicate significant differences from the control (Col-0) using two-tailed t test (*P < 0.05 and**P < 0.01).(F)Expression analysis ofWUSusingGUS staining ofProWUS:GUSPro35S:AtMIF2double transgenicplants treated (upper panel) or not (lower panel) with0.5 mM of TSA. Bars = 10mm.

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hypothesized that their transcriptional regulation should also beconserved and that conserved regulatory elements could well beidentified within the promoters of the two genes. Using in silicoanalysis, we identified several conserved regulatory boxes in theSlIMA and AtMIF2 promoters corresponding to the noncanonicalCArG-box, which is the general target of MADS box proteins(Riechmann et al., 1996). Next, we demonstrated that AG canphysically bind to two of these conserved boxes in vitro by EMSA,and we demonstrated that in vivo, mutation in one of these boxesstrongly reduced the expression of SlIMA.Moreover, the ectopicexpression of TAG1 in tomato led to an increase in SlIMA ex-pression. Altogether, these results demonstrate that AtMIF2 andSlIMA act downstream of AG and TAG1, respectively. Tran-scription factors such as AG are believed to act in a high ordercomplex in which MADS box domain proteins with specificfunctions in organ identity determination are recruited throughtheir interaction with more generic MADS box proteins such asSEPALLATA3 (SEP3) (Kaufmann et al., 2009; Pajoro et al., 2014).The genome-wide interaction of SEP3 with DNA has been ex-tensively studied in various MADS box mutant backgrounds, al-lowing complex, specific downstream targets to be identified(Kaufmann et al., 2009; Pajoro et al., 2014).We therefore surveyedthe published data from AG and SEP3 ChIP-seq experimentsperformed in the wild-type and ag-3 mutant backgrounds. AGChIP-seq experiments did not reveal any binding site inside theAtMIF2 locus sequence (Ó’Maoiléidigh et al., 2013), but SEP3binds to the AtMIF2 promoter in an AG-dependent manner(Kaufmann et al., 2009), suggesting that AG itself or its targetspromote the binding of the MADS box protein complex to theAtMIF2 promoter.

Increased expression of WUS in Arabidopsis results in in-creased floral organ number, particularly locule number (Mayeret al., 1998; Schoof et al., 2000), and silencing of SlWUS in tomatoleads toa reduction in loculenumber (Li et al., 2017).We found thatin AtMIF2/SlIMA-overexpressing and silencing plants, the ex-pression of WUS/SlWUS was downregulated and upregulated,respectively, suggesting that AtMIF2/SlIMA is involved in de-termining locule number through the regulation of WUS/SlWUSexpression. These findings provide evidence that AtMIF2 andSlIMA as representative members of the MIF protein family,functioning as regulatory elements within the AG-WUS pathwaycontrolling FM termination in both Arabidopsis and tomato. Ourresults also point to a general genetic network controlling FMtermination that is conserved between Arabidopsis and tomato.

The AG-KNU-WUS Pathway Is Conserved in Tomato

Previous studies defined the AG-KNU-WUS pathway as impor-tant for regulating floral termination (Sun and Ito, 2015), but theconservation of this pathway in other model plants had beenunclear. Here, we identified the functional homolog of AtKNU intomato, SlKNU, and demonstrated its involvement in repressingSlWUS.Wealsoshowed thatectopic expressionofTAG1stronglyincreasedSlKNUexpression in tomato, indicating thatSlKNUactsdownstream of TAG1. These results provide evidence for theconservation of theAG-KNU-WUSpathway in tomato. The similarregulation of AtMIF2/SlIMA and AtKNU/SlKNU, together with thesimilarity of their partial loss-of-function phenotypes, indicates

thatSlKNUandSlIMAare required to control organnumberduringtomato flower development according to a common molecularregulatory mechanism.

AtMIF2/SlIMA May Form a Chromatin Remodeling ComplexIncluding AtKNU/SlKNU, TPL/SlTPL1, and HDA19/SlHDA1 toRepress WUS/SlWUS

Another interesting question raised by our study is how AtMIF2/SlIMA acts to repress WUS/SlWUS. It has been suggested thatMIFproteins couldact assmall interferingpeptides/microProteinsto inhibit the function of zinc-finger homeodomain (ZHD) proteinsthrough the formation of inactive heterodimers (Hong et al., 2011;Seo et al., 2011; Staudt and Wenkel, 2011; Eguen et al., 2015).Similarly, the C2H2 zinc-finger AtKNU protein harbors an EARtranscriptional repressive motif at its C terminus. As reviewedby Kagale and Rozwadowski (2011), recent discoveries of cor-epressors interacting with EAR motifs, such as TPL, led to theproposal of a model for EAR motif-mediated transcriptional re-pression in plants. In this model, EAR repressors suppress theexpression of target genes through chromatin modification ofregulatory regions by histone deacetylation. TPL, together withHDA19, is thought to form part of a repressor complex that drivesthe epigenetic regulation of gene expression. However, the exactcomposition of the repressor complex remains unknown. In ad-dition, the microprotein, miP1a/b, has been described as anadaptor protein that functions between a zinc-finger transcriptionfactor, CONSTANS, andaTPL-relatedprotein (Graeff et al., 2016).Based on these findings, we wondered whether MIF proteinscould also act as adaptor proteins in such interactions andevaluated the potential interaction between AtKNUand TPL in thepresence of AtMIF2. We demonstrated the required involvementof AtMIF2 and SlIMA as adaptor microproteins engaging AtKNUand SlKNU, respectively, in a ternary complex that involves TPLand HDA19. Our findings confirmed the existence of an adaptorprotein that facilitates the association between TPL and itspartners. We identified this protein as AtMIF2 in Arabidopsis andSlIMA in tomato. We further demonstrated that AtMIF2/SlIMAbind to twodistinct regulatory regions located at21494 to21403and +335 to +489 for AtMIF2, and 21614 to 21424 and +186to +594 in SlIMA (+1 being the transcription start site), to repressWUS/SlWUS in a KNU-dependent manner. These two bindingsitesare localizedatsimilarposition in the twospecies, suggestinga conserved regulatory mechanism. Further work is needed todetermine whether histone deacetylation on the WUS locus oc-curs directly or indirectly to reduce its expression during floraltermination in Arabidopsis and tomato.Mini zinc-finger proteins, which constitute conserved compo-

nents of the floral termination pathway, play key roles in the de-termination of carpel number, and hence fruit locule number, animportant agronomic trait controlling fruit size and yield. Ourresults provide important insights into the conservation of themolecular regulatory network of FM termination in Arabidopsisand tomato. Based on our data, we propose a model integratingMIF2 in the conserved regulatory pathway of FM termination inArabidopsis that can be transposed to tomato (Figure 10). A futurechallenge will be to determine how well conserved the proteininteractions and transcriptional regulation revealed in this study

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are in floral termination events outside of Arabidopsis and tomato.Characterizingsuchconservationorvariationwill providevaluableinsight into the molecular mechanisms underlying the complexregulation of floral stem cell termination.

METHODS

Plant Material and Growth Conditions

Tomato (Solanum lycopersicum cvWest Virginia106-Wva106) plants weregrown in soil in a greenhouse supplemented with light for a 16-h photo-period using a set of 100-W warm white LED projectors. The spectrum isconstituted by equivalent levels of blue irradiation (range 430–450 nm) andred irradiation (640–660nm). The irradiancewas close to100mmolm22 s21

at the canopy level.For transformation, tomato cotyledons were cultivated in vitro in MS

medium (Murashige and Skoog, 1962) in a culture chamber under a ther-moperiod of 22°C/20°C and a photoperiod of 16 h/8 h (day/night) using

white light (Osram L36 W/77 Fluora 1400 Im) resulting in a photon fluxdensity at the stirring plate of 80 to 100 mE m22 s21.

The wild-type Arabidopsis thaliana accessions used in this study areCol-0, Landsberg erecta (Ler), and the cross-product Col-0*Ler. The ag-3mutant (generous gift fromR. Sablowski, John Innes Centre, Norwich, UK)was in the Ler background. Seeds were surface sterilized for 15 min in12.5% (v/v) sodium hypochlorite and 0.02% (v/v) Triton X-100, rinsed atleast five times, and sown in Petri dishes containing 0.53 MS growthmedium for germination. After cold treatment at 4°C for 2 d in the dark, theplates were incubated in a growth chamber at 22°C with a 16-h-light/8-h-dark cycle. After 10 d of growth, the plantlets were transferred to soil ina growth chamber under the same light regime. For TSA treatments,Arabidopsis plantletswere grown in Petri dishes containing 0.53MS in thepresence of 0.5 mM of TSA (Merck). Fourteen-day-old plantlets weremaintained in vitro in the same medium until flowering.

RNA Extraction and qRT-PCR Analysis

Total RNA was isolated from pools of flower buds harvested at differentstagesusingTRIzol reagent (LifeTechnologies) following themanufacturer’sinstructions. RQ1 RNase-free DNase (Promega) treatment was performedfor each sample.Reverse transcriptionwasperformedusing an iScriptOne-Step RT-PCR Kit for Probes (Bio-Rad). For each tissue sample, three bi-ological replicatesandthreetechnical replicatesperbiological replicatewereanalyzed using a CFX96 real-time system (Bio-Rad) and GoTaq qPCRMasterMix (Promega).ForFigures1A,2A,4B,4D,5B,6A,andSupplementalFigure2C, eachbiological replicatewasan independentpoolof tissues from10 plants. For Figures 3B, 5C, and Supplemental Figures 1F and 4, eachbiological replicatewasan independentpoolof tissues fromthreeplants.ForFigures 2B, 4I, 4J, and 6B, each biological replicate corresponded to a poolof tissues from the same plant. The transcript levels of the genes werenormalized to that of the housekeeping genes ELONGATION FACTOR1-a(AT5G60390) and TUBULIN b-2/b-3 chain (AT5G62690) in Arabidopsissamples and SlACTIN2 and SlEiF4e in tomato samples. For the qRT-PCRexperiments, total RNAwas purified from various tomato organs from threeplants using an RNeasy Plant Mini Kit (Qiagen). The RT-PCR experimentsand analyses were performed as described by Joubès et al. (2001). Datawere presented asmean and SD of biological replicates. Supplemental DataSet 1 lists all the primer sequences used in this study.

In Situ Hybridization

In situ hybridizations of mRNA using digoxygenin-UTP-labeled RNAprobeswereperformedasdescribed (Bisbis et al., 2006). To synthesize theriboprobes, specific cDNA fragments were amplified using forwardand reverse oligonucleotides for AtMIF2 (At3G28917) and SlKNU(Supplemental Data Set 1). The cDNA fragments were then amplified bynested PCR using the forward or reverse oligonucleotides (SupplementalData Set 1) to generate the DNAmatrix for antisense and sense riboprobesynthesis, respectively. Antisense or sense RNA probes were obtained bytranscription using T7 RNA polymerase. To be as quantitative as possible,all samples originating from different Arabidopsis or tomato lines wereprocessed in the same way at the same time and prepared on the sameslide for in situ hybridizations.

Production and Purification of Recombinant Protein

To produce AG, AtKNU, and AtMIF2 recombinant proteins, the cDNAfragment encoding each protein was amplified by PCR from cDNAobtained after reverse transcription of RNA extracted from Arabidopsisflowers. Coding sequences were cloned into the pET28a, pET300, andpDEST15expressionvectors togenerate thepET28a-AG,pET300-AtKNU,and pDEST15-AtMIF2 constructs, respectively. These constructs were

Figure 10. Model Illustrating theConservedRole ofMIF2 in theRegulationof FM Termination.

(A) The expression of MIF2 and KNU is activated by AG directly (greenarrows) or indirectly (dashed arrow) in floral buds at stage 3.(B) MIF2 and KNU associate with HDA19 and TPL to form a chromatinremodelingcomplex thatbinds to thefirst intronof theWUS locus, enablingthe complete repression ofWUS (red arrows) and leading consequently tothe termination of stem cell activity at stage 6. se, sepal; pe, petal; st,stamen; ca, carpel.

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then used to transform Escherichia coli BL-21 (DE3) cells to producea 6xHis-tagged version of AG and AtKNU and a GST-tagged version ofAtMIF2. The production of the recombinant protein AG was induced with0.5mM IPTG for 30min at20°C.Under theseconditions, satisfactory levelsof AG were recovered from the soluble protein fraction and subsequentlyused inEMSAexperiments. Theproductionof the recombinantAtMIF2andAtKNU proteins was induced with 0.1 mM IPTG for 16 h at 20°C. Underthese conditions, satisfactory levels of GST-AtMIF2 and 6xHis-AtKNUwere recovered from the soluble protein fraction and subsequently used inpull-down experiments. For purification of 6xHis-tagged protein, bacteriawere lysed for 30 min in a buffer containing 50 mM NaH2PO4 (pH 8.0),300mMNaCl, 10mMimidazole, 1mgmL21of lysozyme, andanEDTA-freeproteinase inhibitor cocktail (Roche; 11836153001) and sonicated fivetimes during a 15-s period. Soluble proteins were then purified usingProtinoNi-NTAAgarose (Macherey-Nagel) according to themanufacturer’sprotocol.

EMSA

The double-stranded probes were 59-end labeled using the T4 poly-nucleotide kinase (Promega) in the presence of [g-32P]-ATP according tothe manufacturer’s instructions. DNA binding assays and gel electro-phoresis were performed as described by Riechmann et al. (1996).

DamID Assay

DamID was performed as described by Germann and Gaudin (2011).Adenine methylation was assayed using a methylation-specific qPCRprotocol (Germann and Gaudin, 2011) with DNA extracted from younginflorescences prior to and after 24 h of ethanol induction (I and NI) in linesexpressingAtMIF2orAtKNUfused toDam(Dam-AtMIF2andDam-AtKNU,respectively) and in lines expressing Dam alone. Extracted DNA was di-gested by DpnII methylation-sensible endonuclease, which cuts onlyGATCs recognition sites where A is not methylated. The DpnII-digestedDNAwasdilutedbeforeuse forqRT-PCR.DamID ratio (DIR)wascalculatedat each GATC site according to Germann and Gaudin (2011).

CRISPR/Cas9 Gene Editing and Genotyping of theResulting Mutants

CRISPR/Cas9 mutagenesis was performed as described (Xu et al., 2015).Briefly, constructswere designed toproducedefineddeletionswithin eachtarget gene-coding sequence using two sgRNAs alongside the Cas9endonuclease gene. The sgRNA target sequences were designed usingCRISPR-P 2.0 web software (http://crispr.hzau.edu.cn/CRISPR2/; Leiet al., 2014) (see Supplemental Data Set 1 for a list of the sgRNAs used inthis study). For genotyping of each first-generation (T0) transgenic line,three different leaf samples were collected, and genomic DNA was ex-tracted using DNAzol (Invitrogen) according to the manufacturer’s in-structions. Each plant was genotyped by PCR for the presence of theconstruct with primers designed to amplify a region spanning the 39 end ofthe construct containing the 39 end of the cas9 sequence and the twosgRNA sequences. The CRISPR/Cas9 T-DNA-positive lines were furthergenotyped for indel mutations using a forward primer to the left of sgRNA1anda reverseprimer to the right of sgRNA2 (SupplementalDataSet 1).PCRproducts fromselectedplantswere purified for cloning into thepDONR201vector usingGateway cloning technology (Invitrogen). Ten clones per PCRproduct were sequenced. Only homozygous plants inwhich all sequencedalleles were mutated were phenotyped to ensure that quantification andcomparison of locule number were based on effectively null mutants. Forquantification of the locule number of the genotyped mutants, we ran-domly collected ;15 fruits from each line and counted the number oflocules. The data from three different mutants were then pooled to

compare with the locule number of wild-type plants grown under thesame conditions.

In Vitro Pull-Down Assay and Immunoblot

Approximately 2 mg of purified prey protein (GST-AtMIF2) was added to6xHis-AtKNU bait protein bound on Protino Ni-NTA agarose beads andincubated for 2 h at 4°C. Six further vigorous washes were performed withNPI-20/wash buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and 20 mMimidazole), and the next elution was performed using NPI-250/elutionbuffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, and 250 mM imidazole).GST-AtMIF2 pulled-down proteins were resolved by 12%SDS-PAGE anddetected by immunoblotting using 1:2000 dilution of mouse monoclonalanti-GST antibody (Santa Cruz Sc-138). A secondary anti-mouse IgG,HRP-linked antibody 7076 (Cell Signaling Technology) was used witha 1:10,000 dilution, and an ECLRevelBlot Intense kit (Ozyme) was used forvisualization of membrane-associated peroxidase activity.

Vector Constructs and Plant Transformation

ProPI:amiRNA-AtMIF2 constructs were produced as follows: the 1735-bpProPI:amiRNA-AtMIF2-t35S sequence (containing 800 bp of the PI pro-moter, 701bpof amiRNA, and222bpof thePro35S terminator surroundedby EcoRI and BamHI restriction sites) was synthesized by Eurofins Ge-nomics into a vector. The entire cassette was cloned into the pPZP212destination vector using EcoRI and BamHI endonucleases.

The IMARNAi constructwasobtained from the IMAcDNAbyamplifyinga 321-bp fragment corresponding to 103 nucleotides from the 39 openreading frame followed by 118 nucleotides of the 39 untranslated region(UTR) using the following oligonucleotides: AAAAAGCAGGCTTGAGA-TATGTTGAGTGCGAG and AGAAAGCTGGGTCACACCTTATTCACA-CACAC. The amplified DNA fragment was cloned using the Gatewaycloning system (Clontech) asdescribedbyKarimi et al. (2002),with pDONR201 as the entrance vector and pK7GWIWG2(1) as the destination vector.

For CRISPR/Cas9 mutagenesis, constructs were designed to createdefined deletions using two sgRNAs. All constructswere assembled usingthe Golden Gate cloning method (Weber et al., 2011). Level 1 constructscarrying sgRNAs placed under the control of the Arabidopsis U6 promoterwere assembled as described (Belhaj et al., 2013). Level 1 constructpICSL11024 (pICH47732:NOSp-NPTII-OCST) was a gift from JonathanD.Jones (Addgene plasmid 51144). Level 1 construct pICH47742:2x35S-59UTR-hCas9(STOP)-NOST was a gift from Sophien Kamoun (Addgeneplasmid 49771). Level 1 constructs pICH47751:AtU6p:sgRNA1,pICH47761:AtU6p:sgRNA2, and the linker pICH41780 were gifts fromSylvestre Marillonnet (Addgene plasmids 48002, 48003, and 48019,respectively). The level 1 constructs were assembled into the level 2vector pAGM4723 (gift from Sylvestre Marillonnet; Addgene plasmid48015) as described (Weber et al., 2011).

For DamID assays, fusions between the AtMIF2 and AtKNU codingsequence and theDamcoding sequencewere produced byPCRusing thepCMycDam vector (thanks to the Van Steensel lab), and the constructswere cloned into apDONRentry vector. Next, these sequencewere clonedinto the pBIN:AlcR vector (provided to us as a generous gift from ValérieGaudin, Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech) inorder to create an ethanol-inducible version of the fusion proteins.

The different constructs were introduced into Agrobacterium tumefa-ciensstrainGV3101pMP90 (KonczandSchell, 1986)usingelectroporation(2.5 kV, 400 liters). Arabidopsis plants were transformed by the floraldip method (Clough and Bent, 1998). After transformation, seeds wereharvested from T0 plants, pooled, and sown on MS medium plates con-taining kanamycin (50 mg mL21) or hygromycin (15 mg mL21), dependingon the transformation vector used. T1 transformed plants were selectedand grown in a growth chamber until mature seeds were obtained. The

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agrotransformation of tomato cotyledons was performed as described(Cortina and Culiáñez-Macià, 2004). Plant regeneration, selection, andelimination of agrobacteria were performed onMSmedium supplementedwith 0.1 mg L21 of IAA, 1 mg L21 of 6-BA, 150 mg L21 of kanamycin, and300mg L21 ofmixed ticarcillin and clavulanic acid (DUCHEFABiochemie).Transformed plants were grown on hormone-free MS medium supple-mentedwith150mgL21of kanamycinand transferred into thegreenhouse.

Yeast Two- and Three-Hybrid Assays

The AtMIF2, SlIMA, TPL, SlTPL1, AtKNU, and SlKNU coding sequenceswere recombined with the pDEST22 and pDEST32 Gateway vectors forfusion with the AD or BD domains, respectively, at their N termini. ThepYES-DEST52 yeast expression vector was used for the expression ofAtMIF2 or SlIMA during the yeast three-hybrid assays.

The empty vectors or vectors containing the respective codingsequenceswere cotransformed into the pJ69-4a yeast strain, and positivecolonies were selected on dropout medium without Trp and Leu for yeasttwo-hybrid analysis or without Trp, Leu, and Ura for yeast three-hybridanalysis. The presence of the plasmids in the strains was verified by PCR,and three positive strains for each transformation were screened on se-lective medium without Trp, Leu, and His for yeast two-hybrid analysis orwithout Trp, Leu, Ura, and His for yeast three-hybrid analysis with addi-tional 75 mM 3-AT. For each experiment, the different possible orienta-tions of the protein fusions were tested to assess interaction, andcotransformationwithemptyplasmidwasperformedasanegative control.

Colocalization and in Cellulo Protein Interaction Assays

Gateway vectors were used for the fusion with YFP- and RFP-taggedproteinsat theN-orC-terminal partsofproteinsof interest.Plasmids for theBiFC experiments were kindly provided by Tsuyoshi Nagakawa (ShimaneUniversity, Japan). Transient expression assays were performed usinga homologous system, namely, leaf epidermis from tomato or Arabidopsis,and onion epidermal cells as a heterologous system. To perform biolistictransformations, slices of onion epidermis were placed on MS medium inPetri dishes. For subsequent bombardment, 2mg of 1.6-mmgold particleswas coated with 5 mg plasmid DNA in the presence of 1 M CaCl2 and150 mM spermidine. Pelleted gold particles were washed consecutivelywith 70% and 100% ethanol and resuspended in 10 mL of 100% ethanolbefore loaded onto macrocarriers for transformation with the particledelivery systemusing an 1100 p.s.i. (7.58MPa) rupture disc (PDS-1000He;Bio-Rad). The distance between the macrocarrier and tissue was 6 cm.After gene delivery, onion, tomato, or Arabidopsis epidermal tissues wereincubatedovernight onMSmediumat roomtemperature in thedarkprior toanalysis. Each transformation assay was performed in triplicate, and eachexperimentwas replicated at least twice.Concerning theBiFCexperiment,all possible orientations of the protein fusions were tested to assess in-teraction. To test spontaneous YFP reconstitution as a negative control,the N- or C-terminal YFP fragment fused to each protein was coexpressedwith the unfused C- or N-terminal YFP fragment and the absence offluorescent signal was assessed (Kudla and Bock, 2016). Supplementalpictures of the BiFC interactions are presented in Supplemental Figure 7.

Microscopy Techniques and Imaging

Images for protein subcellular localization were obtained using the TCSSP2AOBS confocal scanning microscope from Leica. YFP and RFP tags wereexcited and the respective emissions were scanned using a sequential scansetting to prevent overlapping fluorescence signals. All images were pro-cessed using Leica Confocal Software and ImageJ software.

For histological observations, tissues were fixed, embedded in paraffinwax, sectioned, and stained as described (Bereterbide et al., 2002).

GUS staining was performed as described (Mudunkothge and Krizek,2014), observed on paraffin sections under a Zeiss Axioplan microscope,and recorded using a Moticam 3 camera (Motic).

ChIP-qPCR

ChIP assays were performed on young tomato (WVA106 WT andPro35S:SlIMA-YFP) and Arabidopsis (Col-0, Pro35S:AtMIF2-3HA, andPro35S:AtMIF2-3HA in knumutant background) floral buds (stages 1–9)using polyclonal anti-HA (Roche; ref. 11867423001, lot number14553800), anti-GFP (Abcam; ab290, lot numberGR3062151), or controlIgG (Millipore; lot number 2896738) antibodies, using a procedureadapted from Jégu et al. (2013). Briefly, after fixation of the plant materialin 1% (v/v) formaldehyde, tissues were homogenized; nuclei were thenisolated and lysed. Cross-linked chromatin was sonicated in a BioruptorUCD-200 water bath (Diagenode) using the following parameters: 30-s-on/30-s-off pulses, at high intensity for 60 min. Protein/DNA complexeswere immunoprecipitated with antibodies overnight at 4°C with gentleshaking and subsequently incubated for 1 h at 4°C with 50 mL of Dy-nabeads Protein A (100-02D; Invitrogen). Immunoprecipitated DNA wasthen recovered using the ChIP DNA Clean and Concentrator (ZymoResearch). Analiquotof untreatedsonicatedchromatinwasprocessed inparallel for each sample and used as total input DNA control. ConcerningqPCR, for each tissue sample, three biological replicates, each resultingfrom the tissue pools from 48 plants for Arabidopsis samples andfrom five plants for tomato samples, and three technical replicates perbiological replicate were analyzed. For each locus, the fold enrichmentwas calculated by comparing the Ct values of triplicate measurementsbetween immunoprecipitates from transgenic and wild-type plants,relative to the Ct value of the chromatin input control.

Promoter Sequence Analyses

mVISTA software with 70% identity and a sliding window of 20 bases asparameters (Mayor et al., 2000) was used to compare the 2400-bp regionencompassing the tomato SlIMA promoter sequence with the 2400-bpupstream region of Arabidopsis AtMIF2 (At3g28917) in order to identifyconserved regions. The presence of putative transcription factor bindingsites was then analyzed using rVISTA and MatInspector software (Lootsand Ovcharenko, 2004; Cartharius et al., 2005).

Multiple Sequence Alignments and Phylogenetic Analysis

Phylogenetic analysis was performed with C2H2 proteins from Arabi-dopsis and tomato. Multiple sequence alignments were generated viaGeneious software (http://www.geneious.com/) (Supplemental File 1)using BLOSUMmatrix with default parameter setting (gap cost between0.1 and 10). A phylogenetic tree was produced with Geneious TreeBuilder from 1000 bootstrap replicates by applying the neighbor-joiningmethod with Jukes-Cantor-like genetic distance model. Parameter set-tings were the following: no gap penalty, no outgroup, random seed of1000, and support threshold of 10%.

Accession Numbers

All Arabidopsis genes used in this study are referenced in the ArabidopsisGenome Initiative database under the following accession numbers: AG,At4g18960; AtKNU, At5g14010; AtMIF2, At3g28917; AtMIF1, At1g74660;AtMIF3, At1g18835; WUS, At2g17950; TPL, At1g15750; HDA19,At4g38130;ACTIN2 (ACT),At3g18780;andEF1a (EF1),At5g60390.Tomatosequence information can be found in the Sol Genomics Network (http://solgenomics.net;Fernandez-Pozoetal.,2015)under thefollowingIDs:TAG1,Solyc02g071730; SlKNU, Solyc02g094428; SlWUS, Solyc02g083950;

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SlIMA, Solyc02g087970; SlTPL1, Solyc01g100050; and SlHDA1,Solyc09g091440.

Supplemental Data

Supplemental Figure 1. Expression of the ProPI:GUS reporter geneand floral phenotypes of Arabidopsis ProPI:amiRNA-AtMIF2 lines.

Supplemental Figure 2. SlKNUCKLES phylogenetic and expressionanalyses of SlKNUCKLES.

Supplemental Figure 3. The SlIMA and AtMIF2 promoters containhighly conserved sequences.

Supplemental Figure 4. Expression analysis of TAG1 in the wildtype, ProSlIMA:GUS, and Pro35S:TAG1-overexpressing line usingqRT-PCR.

Supplemental Figure 5. Subcellular localization of the proteinsstudied.

Supplemental Figure 6. Characterization of the interaction domaininvolved in the interaction between IMA/MIF2 and KNU/SlKNU.

Supplemental Figure 7. Additional BiFC analysis

Supplemental Figure 8. BiFC analysis of the interactions betweenAtKNU and TPL and between SlKNU and SlTPL1 in the presence oftagRFP in onion epidermal cells.

Supplemental Data Set 1. List of primer sequences used in thisstudy.

Supplemental File 1. Alignment used to produce the phylogenetictree shown in Supplemental Figure 2.

ACKNOWLEDGMENTS

We thank Valérie Gaudin for valuable advice and protocols on DamID.We thank Catherine Perrot-Rechenmann and Thierry Desnos for thepbinSRNACatN plasmid. The microscopy was done in the BordeauxImaging Center, Plant Imaging Plateform, UMS 3420, INRA-CNRS-INSERM-University of Bordeaux, member of the national infrastructureFrance BioImaging. This work was financed by a grant from the Ministèrede l’Enseignement Supérieur et de la Recherche (for N.B., PhD).

AUTHOR CONTRIBUTIONS

F.D., N.B., A.S., C.C., and M.H. conceived the project and designed theresearch. N.B., A.S., J.L., F.D., and M.H. conducted the experiments. A.S.performed EMSA and rVISTA analyses. N.B., F.D., andM.H. produced thetransgenic plant material. N.B., D.L., M.B., and C.R. contributed to ChIPstudies. N.G., F.G., and M.L. helped analyze the results. All authorsdiscussed the results. N.B., N.G., C.C., M.H., and F.D. wrote the manu-script with input from the other authors.

Received August 28, 2017; revised November 8, 2017; accepted Decem-ber 20, 2017; published January 3, 2018.

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100 The Plant Cell

DOI 10.1105/tpc.17.00653; originally published online January 3, 2018; 2018;30;83-100Plant CellFrédéric Delmas

Moussa Benhamed, Cécile