2409RESEARCH ARTICLE

INTRODUCTIONUnlike animals, which can initiate germlines as early asembryogenesis, plant reproductive cells form late in development.All male reproductive cells of Arabidopsis develop withinconcentrically organised microsporangia derived from archesporialcells in the L2 of the stamen primordium (Fig. 1A,E). Thesearchesporial cells divide periclinally to form an inner primarysporogenous cell and an outer primary parietal cell (Fig. 1B,E).Primary sporogenous cells continue division to give rise to a centralsporogenous mass, and consecutive divisions of neighbouring cellsresult in the formation of three concentric parietal layers – thetapetum adjacent to the sporogenous cells, a middle layer, and theendothecium subjacent to the epidermis (Fig. 1C-E) (Canales et al.,2002; Feng and Dickinson, 2007; Scott et al., 2004; Sorensen et al.,2002). In common with the development of other plantreproductive organs, where consistency in size and shape isessential for effective function, microsporangial formation does notinvolve ‘classical’ plant meristems, but rather finite numbers ofdivisions within apparent cell lineages – reminiscent ofdevelopment in some animal tissues.

Patterning of the concentric microsporangia involves theinterplay of many genes (Feng and Dickinson, 2007; Ma, 2005;Wilson and Yang, 2004). The transcription factorSPOROCYTELESS (SPL; also known as NOZZLE, NZZ)(Schiefthaler et al., 1999; Yang et al., 1999) acts directly

downstream of the floral ‘C’ gene AGAMOUS (AG) (Ito et al.,2004) to promote sporogenous cell identity (Schiefthaler et al.,1999; Yang et al., 1999) and is reported to interact with the leucine-rich repeat receptor-like kinases (LRR-RLKs) BARELY ANYMERISTEM 1 and 2 (BAM1 and BAM2) (Hord et al., 2006).BAM1 and BAM2 are held to promote somatic cell fates, first inthe outward-facing products of the archesporial cell division, andsubsequently during microsporangial expansion by restricting theexpression domain of SPL to the locular centre (Hord et al., 2006).In turn, SPL appears to promote BAM1 expression, possiblycreating a regulatory loop similar to that involving CLAVATA 1(CLV1) and WUSCHEL (WUS) in shoot apical and floral meristems(Hord et al., 2006).

Also playing an important part in microsporangial patterning, theLRR-RLK EXCESS MICROSPOROCYTES 1 (EMS1; alsoknown as EXTRA SPOROGENOUS CELLS, EXS) (Canales etal., 2002; Zhao et al., 2002) is reported to form a receptor complexwith two further LRR-RLKs – SOMATIC EMBRYOGENESISRECEPTOR-LIKE KINASE1 and 2 (SERK1 and SERK2) – in theplasma membrane of tapetal cells (Albrecht et al., 2005; Colcombetet al., 2005). This complex is held to bind TAPETUMDETERMINANT 1 (TPD1) (Yang et al., 2005; Yang et al., 2003),a putative ligand synthesised by the microsporocyte mass, and isreported to specify tapetal cell fate (Albrecht et al., 2005; Feng andDickinson, 2007; Jia et al., 2008; Ma, 2005; Yang et al., 2005).ems1 microsporangia have no tapetal layer but excessmicrosporocytes (Canales et al., 2002; Zhao et al., 2002), aphenotype shared with serk1 serk2 double mutant lines (Albrechtet al., 2005; Colcombet et al., 2005) and tpd1 plants (Yang et al.,2003). How this EMS1 complex mediates the formation of afunctional tapetal layer is unknown; models range from a role inregulating the number of functional archesporial cells (Canales et

Development 137, 2409-2416 (2010) doi:10.1242/dev.049320© 2010. Published by The Company of Biologists Ltd

Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX13RB, UK.

*Author for correspondence (hugh.dickinson@plants.ox.ac.uk)

Accepted 14 May 2010

SUMMARYThe four microsporangia of the flowering plant anther develop from archesporial cells in the L2 of the primordium. Within eachmicrosporangium, developing microsporocytes are surrounded by concentric monolayers of tapetal, middle layer and endothecialcells. How this intricate array of tissues, each containing relatively few cells, is established in an organ possessing no formalmeristems is poorly understood. We describe here the pivotal role of the LRR receptor kinase EXCESS MICROSPOROCYTES 1(EMS1) in forming the monolayer of tapetal nurse cells in Arabidopsis. Unusually for plants, tapetal cells are specified very early indevelopment, and are subsequently stimulated to proliferate by a receptor-like kinase (RLK) complex that includes EMS1.Mutations in members of this EMS1 signalling complex and its putative ligand result in male-sterile plants in which tapetal initialsfail to proliferate. Surprisingly, these cells continue to develop, isolated at the locular periphery. Mutant and wild-typemicrosporangia expand at similar rates and the ‘tapetal’ space at the periphery of mutant locules becomes occupied bymicrosporocytes. However, induction of late expression of EMS1 in the few tapetal initials in ems1 plants results in theirproliferation to generate a functional tapetum, and this proliferation suppresses microsporocyte number. Our experiments alsoshow that integrity of the tapetal monolayer is crucial for the maintenance of the polarity of divisions within it. This unexpectedautonomy of the tapetal ‘lineage’ is discussed in the context of tissue development in complex plant organs, where constancy insize, shape and cell number is crucial.

KEY WORDS: Anther tapetum, Arabidopsis, Cell fate establishment, EMS1, Reproductive cell lineage

Tapetal cell fate, lineage and proliferation in the ArabidopsisantherXiaoqi Feng and Hugh G. Dickinson*

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al., 2002) to the specification of tapetal fate in the inner divisionproducts of the inner secondary parietal cells, cells which, in ems1mutant lines, are held to revert to a default microsporocyte fate –implying a switch in fate within a cell lineage (Zhao et al., 2002).

In an attempt to define further the role of the EMS1 complex inthe specification of the Arabidopsis tapetum and the mechanism bywhich it proliferates as a monolayer, we expressed EMS1ectopically in ems1 plants after its normal period of expression.These experiments show that a small population of founder cells iscommitted to a tapetal fate very early in development, but that theEMS1 complex is not involved in this early specification.However, the EMS1 complex is required for the subsequentproliferation of these cells to form the tapetal monolayer, but notdivisions in any other cell layer. We also show competition forspace to exist between the products of the microsporocyte andtapetal cell lineages. This early independence of the tapetal lineageand its ability to interact with neighboring cell layers provide thefirst clues as to how these small but complex plant organs develop– apparently in the absence of functional meristems.

MATERIALS AND METHODSPlant materials and growth conditionsWild-type (wt) Arabidopsis thaliana (Linneaus) Landsberg erecta (Ler)plants were used unless otherwise specified. exs1, exs2, exs3 seeds wereavailable in the laboratory (Canales et al., 2002). ems1 seeds were kindlyprovided by Hong Ma (Pennsylvania State University, USA) (Zhao et al.,2002); tpd1 seeds by De Ye (China Agricultural University, China) (Yanget al., 2003); and serk1 serk2 seeds by Sacco de Vries (WageningenUniversity, Holland) (Albrecht et al., 2005). Rod Scott (Bath University,UK) provided pA9::Barnase lines in both Ler and Columbia-0backgrounds (Dilkes et al., 2008). The ems1 line was genotyped usingXF301, XF504 and XF3R primers, and tpd1 (Yang et al., 2003) and serk1serk2 (Albrecht et al., 2005) mutants were genotyped as previouslydescribed. Plants were grown at 21°C on a 16 hour light/8 hour dark cycle.

RT-PCR analysisBiological replicates comprised floral buds at flower stages (Smyth et al.,1990) 1-14, collected from three plants, and three independent replicateswere used from each line. Quantitative RT-PCR (qRT-PCR) was performedwith an Applied Biosystems 7300 Real-Time PCR system, usingXG102F/XG102R primers for AtA9 and At/AhEF1a primers (Becher et al.,2004) as a control. Thirty-five cycles of RT-PCR were carried out(PyrobestDNA polymerase, TaKaRa) using XG1206F/XG1206R primersfor EMS1 and GAPDH primers (Hay et al., 2002) as a control.

Transgenic plants4005 bp of EMS1 genomic DNA and 979 bp of the A9 promoter (pA9)were cloned using primers XF201/XF202, and XF203/XF204, respectively.The pA9::EMS1 fragment was amplified through interlaced PCR usingprimers XF201/XF204. This fragment was cloned into pMLBART (Gleave,1992) using NotI digestion. The same fragment was also cloned intoGateway pDONR207 vector (Invitrogen) after amplification using primersXL101/XL102, and finally recombined into Gateway vector pGWB16(Invitrogen). Both constructs were then introduced into ems1/+heterozygous plants using floral dip transformation (Zhang et al., 2006).The pMLBART-pA9::EMS1 and pGWB16-pA9::EMS1-6xcMYC T1 plantsresulting were screened for BASTA, and for Kanamycin and Hygromycinresistance, respectively, and were genotyped for the transgene usingXH201/XH203 primers and for the genomic EMS1 allele usingXF301/XF504/XF3R primers. T4 plants from each independent transgenicline were genotyped and examined for phenotype.

Determination of stage specificity of the AtA9 promoterInterpretation of our experiments required precise knowledge of the timingof pA9 expression in our plant lines; previous reports had only describedpA9 driven reporter constructs as being tapetal specific (Sorensen et al.,2002). We thus investigated tapetal ablation in pA9::Barnase expressing

lines of Arabidopsis (Dilkes et al., 2008; Paul et al., 1992). These plantsform a full complement of microsporocytes and tapetal cells, but the latterbecome highly vacuolated during the proliferation phase (S5; see Fig. S1in the supplementary material), expand rapidly and degenerate after S5,confirming pA9 to be tissue specific and active during the proliferation ofthe tapetum.

Light microscopy and measurementsMaterial was prepared as previously described (Sorensen et al., 2002),observed under an Olympus BX50 microscope and imaged using Image-Pro 6.2 software. The longitudinal mid-point of abaxial microsporangiafrom the medial anthers of anther stage 6 was chosen for cell numbermeasurement. Mean value and s.d. were calculated from 150 independentlocules of each line.

Transmission electron microscopyMaterial was prepared as previously described (Sorensen et al., 2002).

In situ hybridisationA 1157-bp fragment for EMS1 detection was amplified using primersXH402F/XH402R (Zhao et al., 2002) using wild-type cDNA as template;similarly, a 305-bp A9 fragment was amplified using primersXH401F/XH401R, and a fragment for A7 detection was made using DNAfrom vector pMC1577 kindly provided by Hong Ma (Zhao et al., 2002;Rubinelli et al., 1998). Fragments were cloned into the pGEM T-Easyvector (Promega) and checked by sequencing. In situ hybridisation wascarried out on 8-mm paraffin wax-embedded sections using antisense andsense probes for each gene as previously described (Lincoln et al., 1994).

PrimersSee Table S1 in the supplementary material for a list of primers used.

RESULTSTapetal initials in the Arabidopsis anther arespecified early, and require the EMS1 receptorkinase to proliferateTo investigate the role of the EMS1 complex in tapetaldevelopment, we first examined events immediately following thedivision of the archesporial cell in wild-type Arabidopsis (Fig. 1A-I). At this stage, and only in the region under the radius of theanther lobe (distant from the connective), the primary parietal celland its adjacent two or three L2 cells (in transverse section)surrounding the primary sporogenous cell divide to form outer, andinner, secondary parietal cells (Fig. 1C). The primary sporogenouscells commence division at this point and, simultaneously, the innersecondary partietal cells adjacent to them divide periclinally to giverise to tapetal and middle cell layer initials, which we term primarytapetal cells (PTCs) and primary middle layer cells, respectively.This results in the sporogenous cells becoming surrounded by asmall number of PTCs (Fig. 1F). Dramatic growth of themicrosporangium then follows, achieved principally by cellexpansion and anticlinal division of the investing layers, and oneor two further, apparently unpolarised, divisions of the sporogenouscells. The PTCs participate actively in this proliferation throughsuccessive anticlinal divisions, maintaining a monolayer of cellsbetween the microsporocyte mass and the dividing middle layer(Fig. 1G). The microsporocytes then enter meiosis and producetetrads (Fig. 1H) from which individual microspores are released(Fig. 1I) to develop, via pollen mitosis 1 and 2, into pollen grains.

ems1 microsporangia develop similarly to wild type prior toanther stage 5 [S5, anther stages refer to Sanders et al. (Sanders etal., 1999) (Fig. 1J)]; however, the PTCs in these lines fail toproliferate, and as both sporogenous cells (on the inside), and theouter layers of the microsporangium (on the outside) continue todivide and expand, individual PTCs either remain grouped (Fig.

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1K,L), or become separated from each other, isolated at the locularperiphery (Fig. 1M,N). As these cells do not develop intofunctional tapeta in ems1 locules, we have termed them locularperipheral cells (LPCs) from this stage onwards. On average, eachems1 microsporangium contains 3.15±0.34 LPCs in transversesection at the mid-point of the abaxial microsporangium of medialanthers at anther stage 6. The locular content of ems1 plantsdegenerates shortly following the second meiotic nuclear division(Canales et al., 2002; Zhao et al., 2002), with LPCs showingdramatic hypertrophy (Fig. 1L,N).

The locular peripheral cells of ems1microsporangia express tapetal cell markersTo determine what ‘tapetal’ features are possessed by the LPCs inthe ems1 mutants, we examined these cells using transmissionelectron microscopy (TEM) and investigated the expression of twotapetal markers, At5g07230 (A9) expressed early in development(Paul et al., 1992) and At4g28395 (A7) expressed post meiosis(Rubinelli et al., 1998). TEM analysis showed the LPCs to featurea high density of ribosomes, and active populations of Golgi bodiesand mitochondria (see Fig. S2 in the supplementary material) – allfeatures of tapetal cells. Quantitative RT-PCR (qRT-PCR) showsA9 to be expressed in wild-type and ems1 plants – but at a lowerlevel than in the mutant (see Fig. S3 in the supplementarymaterial). In situ hybridisation confirms the presence of A9transcript in the developing tapetal cells of wild type (Fig. 2A-D),

as well as solely in the LPCs of ems1 anthers (Fig. 2E-H). A7 ishighly expressed in the active, late tapetal cells of wild-type anthers(see Fig. S4A in the supplementary material) and, although theloculi of ems1 plants degenerate after meiosis 2 (Canales et al.,2002), the A7 transcript can nevertheless be detected in thehypertrophic LPCs of these anthers using in situ hybridisation (seeFig. S4C,D in the supplementary material). A combination ofposition and marker gene expression thus clearly identifies theLPCs of ems1 locules as partially developed tapetal cells.

We investigated the possibility that the LPCs in ems1 plantsresult from low levels of normal or aberrant EMS1 expression bysearching for functional EMS1 transcripts in ems1 plants using RT-PCR (see Fig. S5 in the supplementary material). No transcriptswere detected, and further evidence that the LPCs do not resultfrom incomplete penetrance of the ems1 mutation is provided bythe presence of identical numbers of morphologically similar LPCsin exs-1, and exs-2 and exs-3 mutants in the C24 ecotype (data notshown).

Ectopic expression of the EMS1 receptor kinase inthe locular peripheral cells of ems1 plantsstimulates their proliferation and restores tapetalfunctionTo discover whether the few LPCs present in ems1 microsporangiacan be ‘reactivated’ to generate functional tapetal cells (mimickingwild-type development), we expressed EMS1 in ems1 plants under

2411RESEARCH ARTICLETapetal development in Arabidopsis

Fig. 1. Development of Arabidopsismicrosporangia from L2 archesporial initials.Cross sections of anthers at different developmentalstages (anther stages S2-S9). (A-D)Wild-type (WT)microsporangial pattern formation through celldivisions. (E) Cell division events. The cell lineagegiving rise to reproductive cells is highlighted in pink.(F-N)Microsporangial development in WT (F-I) andems1 mutant (J-N). ems1 and WT development issimilar (F,J) until S4 when ISP cell division gives rise toPTCs and PMLCs. In WT, these proliferate anddevelop into tapetum and middle cell layer (G). Inems1 mutants, PTCs fail to divide and differentiate.Instead these cells (hereafter termed LPCs) remain atthe periphery of the locule (red arrowheads), whileMSCs continue to divide (K,M). At S6, WT MSCsenter meiosis to produce tetrads (H); these releasemicrospores (I). Tetrads degenerate (L,N) in ems1 linesand LPCs (red arrowheads) become enlarged andvacuolate. In all figures: Ar, archesporial cell; En,endothecium; ISP, inner secondary parietal cell; LPC,locular peripheral cell; M, middle cell layer; MSC,microsporocyte; Msp, microspore; OSP, outersecondary parietal cell; PMLC, primary middle layercell; PP, primary parietal cell; PS, primary sporogenouscell; PTC, primary tapetal cell; Sp, sporogenous cell; T,tapetum; Td, tetrad; WT, wild type. S denotes antherstages (Sanders et al., 1999). Scale bars: 20mm.

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the A9 promoter (pA9), which drives tapetal-specific expressionduring the proliferation stage of the tapetum (see Materials andmethods) and which, in ems1 locules, is expressed only in theLPCs. In all 42 ems1 pA9::EMS1 and pA9::EMS1-cMYC linesgenerated, LPCs both proliferated and differentiated into apparentlynormal tapetal cells (see Fig. 3) – to which the EMS1 protein wasrestricted (data not shown). All lines produced fertile pollen,despite the comparatively late expression of EMS1 when driven bypA9 [EMS1 expression precedes that of A9 in the wild-type anther(Canales et al., 2002; Paul et al., 1992; Zhao et al., 2002)] (Fig. 2A-D,I-L), but each transgenic line possessed a different proportion ofsterile locules. The tapetum was also restored in different patternsin different lines, varying from a normal monolayer (e.g. in ems1pA9::EMS1 line #10; Fig. 3A-C) to clumps of multilayeredtapetum (e.g. in ems1 pA9::EMS1 line #5; Fig. 3D-F).

pA9::EMS1 expression in ems1 microsporangia restores tapetalfunction sufficiently to produce viable pollen and, because only theLPCs express A9 in these locules, it follows that the functionaltapeta of the ‘restored’ plants must be derived from this cell type.Furthermore, as the tapetum of some of the transgenic lines (e.g.ems1 pA9::EMS1 line #10) so closely resembles that of wild type,and because the LPCs themselves resemble tapetal cells both inposition and in expression of the A9 (early) and A7 (late) tapetalmarkers, we conclude that the wild-type tapetal layer must also bederived from the few PTCs formed around the early sporogenouscells. The extra tapetal cells that are frequently generated in ems1pA9::EMS1 lines are likely to be the result of the higher activity ofpA9 compared with pEMS1 (Fig. 2I-P).

Although pA9 is activated after EMS1 transcription in wild-typeplants (Canales et al., 2002; Paul et al., 1992; Zhao et al., 2002)(Fig. 2A-D,I-L), it must direct expression sufficiently early for thetransgenic tapeta to follow closely the ‘wild-type’ developmentalpathway. pA9::EMS1 rescues the ems1 mutant phenotype, but itfails to do so in either ems1 tpd1 double or ems1 serk1 serk2 triplemutant backgrounds, although EMS1 is strongly expressed in theLPCs of the mutant anthers (see Fig. S6 in the supplementarymaterial). However, these transgenic plants have phenotypes verysimilar to those of ems1, tpd1 and serk1 serk2 mutants (Fig. 4),providing further evidence that these four genes are members of thesame signalling pathway (Albrecht et al., 2005; Colcombet et al.,2005).

Inducing primary tapetal cells to proliferate intransgenic plants restricts the microsporocytepopulationAs the reduction in tapetal cell number in ems1 lines isaccompanied by an increase in the microsporocyte population, weinvestigated the relationship between microsporocyte number andthe level of tapetal proliferation in ems1 pA9::EMS1 transgenicplants. In wild-type plants, mid-point transverse sections ofmedial anthers with microsporangia at stage 6 transect 7.17±0.17microsporocytes, whereas similar ems1 locules contain14.38±0.32 microsporocytes. The number of microsporocytes inems1 pA9::EMS1 microsporangia varies markedly between lines,but they are always in excess of those in wild-type plants, andless than those found in ems1 anthers. For example, ems1

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Fig. 2. A9 is expressed in ems1 LPCs, and the A9promoter drives high levels of EMS1 expressionin ems1 pA9::EMS1 tapeta. (A-T)In situhybridisations showing anthers at differentdevelopmental stages. (A-H)A9 transcript in WT(A-D) and ems1 mutant (E-H). A9 expressioncommences from S5: exclusively within the tapetum(C and D; green arrows) in WT, or in the LPCs (G andH; red arrows) of the ems1 mutant. (I-P)EMS1transcript in WT (I-L) and ems1 pA9::EMS1 plants(M-P; ems1 pA9::EMS1 line #5). In WT, EMS1 isexpressed in all cells of the microsporangium beforeS5 (I), and accumulates strongly in the tapetum(green arrows) from S5 onwards (J-L). In ems1pA9::EMS1 plants, EMS1 is only induced in thetapetum (N-P; green arrows), but at a greater levelthan when driven by its own promoter in WT.(Q-T)Control hybridisations with sense probes on WT(Q,R) and ems1 pA9::EMS1 (S,T) anthers at stages 4(Q,S) and 11 (R,T). The background signal generatedby developing microspores is evident. Scale bars:20mm.

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pA9::EMS1 line #5 sporangia contain 10.38±0.18microsporocytes in transverse section (see Fig. S7 in thesupplementary material). Similar comparisons cannot be carriedout for tapetal cells as the number in ems1 pA9::EMS1microsporangia cannot be measured accurately owing to theirmultiple division planes and irregularity in size and shape.However, the fact that ems1 anthers (which contain excessmicrosporocytes) contain few LPCs (about three per section),transgenic ems1 pA9::EMS1 lines (which contain fewermicrosporocytes than ems1 plants) have significant numbers oftapetal cells, and wild-type anthers (which possess fewermicrosporocytes than ems1 pA9::EMS1 lines) feature a completetapetal monolayer, points to a clear inverse relationship betweentapetal cell and microsporocyte numbers.

However, even when a functional monolayer of tapetal cells isformed in ems1 pA9::EMS1 lines (e.g. as in ems1 pA9::EMS1 line#10), the number of microsporocytes always exceeds that of wild-type anthers. pA9 is activated later in anther development thanpEMS1, and this interval between expression stages may provide anopportunity for the microsporocytes to commence proliferation in theabsence of a developing tapetal layer. Significantly, in all the ems1and pA9::EMS1 ems1 lines screened, the numbers of cellscomposing the middle, endothecial and epidermal layers wereidentical to those of wild-type plants, showing that the proliferationof these cell layers is unaffected by the presence or absence of bothEMS1 expression and a functional tapetal layer.

Many ems1 pA9::EMS1 microsporangia degenerate after S9(Fig. 3F), and we found a clear relationship betweenmicrosporocyte number, the extent of tapetal disruption and locularfertility. Lines with a large number of microsporocytes were likelyto possess more fragmented and multilayered tapetum, and a higherproportion of sterile locules. For example, ems1 pA9::EMS1 line#5, which contains 10.38±0.18 microsporocytes in transversesection (the highest number among all 24 transgenic lines), has themost irregular tapetum and the largest number of sterile locules(see Fig. S7 in the supplementary material).

Tapetum derived from isolated locular peripheralcells proliferates via periclinal and anticlinaldivisions in transgenic plantsOur screen of microsporangia from different transgenic linesrevealed a wide range of phenotypes, including plants containingclumped or multilayered tapetum (Fig. 3D-F). We believe this, likethe higher numbers of microsporocytes (see above), to result fromdifferences in the timing of EMS1 function (Fig. 5). Thus, indifferent ems1 pA9::EMS1 lines, the promoter might drive EMS1transcription to attain an effective threshold either before or afterthe fragmentation of the monolayer of LPCs around thesporogenous cells: if before, exclusively anticlinal division takesplace; if afterwards, a mixture of anti- and periclinal divisionoccurs to generate tapetal layering (Fig. 5). The associationbetween the integrity of the tapetal monolayer and division polarityis striking, and points to the polarity of division (anticlinal in thiscase) being maintained by coordinating signals from neighbouringcells. Such signals might be transmitted through plasmodesmata,or generated by mechanical stress set up in the tapetal monolayeritself. Alternatively, division polarity could be regulated by thedirectionality of the TPD1 signal from the microsporocytes. Inwild-type locules, this signal would be received on the inward

2413RESEARCH ARTICLETapetal development in Arabidopsis

Fig. 3. pA9::EMS1 restores functional tapetum to ems1 mutantanthers. Light micrographs of sectioned anthers of ems1 pA9::EMS1plants. Rescued tapetal cells form a continuous layer (A-C), or multiplelayers (D-F) that are sometimes discontinuous (E,F). Monolayered tapetawith near-normal MSC numbers are fertile (e.g. ems1 pA9::EMS1 line #10;A-C); excess tapetal cells result in degeneration (e.g. ems1 pA9::EMS1 line#5, D-F). Green arrows indicate tapetum. Scale bars: 20mm.

Fig. 4. pA9::EMS1 does not restore functional tapetum to ems1tpd1 or ems1 serk1 serk2 mutant lines. (A-L)Light micrographs ofsectioned anthers. (A-C)tpd1 mutant microsporangia. (D-F)ems1 tpd1pA9::EMS1 microsporangia. (G-I)serk1 serk2 microsporangia. (J-L)ems1serk1 serk2 pA9::EMS1 microsporangia. pA9::EMS1 rescues tapetum inems1 but not in tpd1 or serk1 serk2 mutant backgrounds; these showphenotypes identical to the those of the original mutant lines (tpd1,A-C; serk1 serk2, G-I), where PTCs (red arrows) are present either as agroup (A,D,G,J) or individual cells (B,E,H,K). Scale bars: 20mm.

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facing surfaces of the tapetal cells, whereas in ems1 pA9::EMS1lines, where LPCs are isolated, the TPD1 signal could be perceivedon their radial (and perhaps outward) surfaces. Such a system hasbeen reported for stomatal development, where the LRR-RLKsERECTA (ER), ERECTA LIKE 1 (ERL1) (Shpak et al., 2005) andTOO MANY MOUTHS (TMM) (Geisler et al., 2000; Nadeau andSack, 2002) form a heterodimer, which orients the cell divisionplate depending upon from which direction it receives the ligandEPIDERMAL PATTERNING FACTOR 1 (EPF1) (De Smet et al.,2009; Hara et al., 2007).

DISCUSSIONThe sequence of cell divisions leading to tapetal formation is wellunderstood in Arabidopsis (Canales et al., 2002; Feng andDickinson, 2007; Scott et al., 2004), but how cells generated by thepericlinal division of the inner secondary parietal (ISP) cell layeracquire and maintain their tapetal fate is unclear. The similarity incell numbers between wild type and ems1 mutants (where notapetum is formed) has reasonably been interpreted as evidence thatthese ISP cells revert to a default microsporocyte fate in theabsence of the EMS1 receptor kinase (Zhao et al., 2002). However,our findings that a small number of cells with tapetal features(PTCs/LPCs) are formed early in development in wild-type and

ems1 plants, and that ectopically expressed EMS1 stimulatesproliferation of only these cells in the latter indicate that analternative interpretation of these events might be required.

The specification, proliferation and maintenanceof the Arabidopsis tapetal cell lineageThe new data point to the small number of cells formed adjacentto the sporogenous cells immediately becoming committed to a‘pre-tapetal’ fate, and the EMS1 signalling pathway beingresponsible for the subsequent proliferation of these cells, allowingthe tapetal monolayer to keep pace with the rapid expansion of theother layers of the loculus, which, because they are unaffected bythe absence of functional EMS1, must be regulated by differentsignals. This role for EMS1 in proliferation implies that the EMS1complex cannot be involved in the initial establishment of tapetalfate in the PTCs; the genes responsible for this are unknown, butother LRR-RLKs, such as BAM1/2, remain possible candidates.

Arguably, the LPCs of ems1 anthers could represent a smallnumber of partially developed tapetal cells resulting from theincomplete penetration of the ems1 mutation, with the remainderof the inner division products of the inner secondary parietal cellbeing converted into microsporocytes. This is unlikely because: (1)EMS1 transcripts are absent from ems1 mutant plants; (2)

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Fig. 5. Model for wild-type, ems1 and ems1 pA9::EMS1 microsporangial development. Division of ISPs occurs between S3 and S4 togenerate PTCs and PMLCs. In WT PTCs, the EMS1 signalling complex becomes active, stimulating proliferation. In ems1, the complex is inactive andPTCs fail to divide, instead they differentiate further as LPCs. The MSCs and PMLCs divide as the locule expands in ems1, fragmenting the LPC layer.Expression of the pA9::EMS1 transgene activates the EMS1 signalling complex later in development: if this occurs before fragmentation of the LPClayer, a tapetal monolayer is formed (as in ems1 pA9::EMS1 line #10); if afterwards, clumps of cells resulting from peri- and anticlinal division areformed (as in ems1 pA9::EMS1 line #5). The later the EMS1 functional threshold is achieved, the more time MSCs have to proliferate beforeinhibition by tapetal cells – either through physical competition for locular space or by signalling.

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reactivation of these few LPCs in ems1 lines can generate acomplete and functional tapetum that supports the production ofviable pollen; and (3) LPCs are present in all plants carrying ems1alleles in both Ler and C24 backgrounds (some encoding non-functional receptor kinases), as well as in tpd1 and serk1 serk2plants.

Although the EMS1 complex is required for the proliferation ofPTCs, its role in their final differentiation into functional tapetalcells is unclear. LPCs certainly complete some aspects of tapetaldevelopment in ems1 lines as they express A7, a gene encoding alipid transfer protein that is normally expressed post meiosis(Rubinelli et al., 1998), but they nevertheless fail to develop intofunctional tapetal cells. The EMS1 complex and its putative ligandTPD1 thus emerge as a component of a larger gene networkregulating tapetal development, with other network members beingresponsible for specifying initial tapetal fate in the PTCs and,perhaps, aspects of subsequent development. Although an inverserelationship exists between microsporocyte and tapetal cellnumbers in our transgenic plants, our data do not show whether thisis the result of intercellular signalling, or simply the proliferationof reproductive cells to fill space that would otherwise be occupiedby the tapetal monolayer.

The formation of the tapetal monolayer duringmicrosporangial developmentOur experiments begin to explain how complex tissue arrays candevelop within small plant organs in the absence of organisedmeristems. Like the microsporocytes, tapetal cells are specifiedvery early, and once specified their fate cannot be altered. Thesetapetal ‘founders’ are then stimulated to proliferate (via the EMS1complex) resulting in the formation of a monolayer investing themicrosporocyte population. This signal for proliferation is uniqueto the tapetum, and differs from that regulating division of themicrosporocytes and the other cell layers of the microsporangium.As TPD1 binds to the EMS1 complex (Yang et al., 2005), and isprincipally synthesised by the microsporocytes (Yang et al., 2003),its presence must serve as a developmental checkpoint confirmingthe presence of functional microsporocytes (Fig. 6). Maintenance

of the developing monolayer requires its continued integrity,revealing that anticlinal divisions within it are either controlled byinternal signals, or by microsporocyte-generated TPD1 acting as adirectional cue. Cells of the tapetum and the microsporocytes thusdevelop as ‘communicating’ neighbours, but as independentlineages occupying defined domains within the expandingmicrosporangium. Whether the proliferation of microsporocytesinto ‘tapetal territory’ in ems1 lines results from the lack of signalsfrom the tapetum, or simply from available space, remains to bedetermined.

Perhaps the most significant difference between development ofthese complex microsporangial tissues and development of thosederived from the apical and root meristems is their early andcontinued independence; certainly they can receive both directionaland ‘checkpoint’ signals and perhaps alter the space they occupybut, once formed, their founder cells generate independentlyproliferating lineages committed to single developmental fates.Without this degree of independence and level of ‘internal’ controlit is difficult to see how small numbers of cells of different fatescan assemble into such a range of intricate patterns within a small,terminally differentiated organ such as the anther.

AcknowledgementsWe thank the following for providing mutant lines and reagents: Hong Ma, DeYe, Sacco De Vries, and Rod Scott for providing the pA9::Barnase lines andinformation on A9 expression patterns. Carla Galinha and Paolo Piazza gavevaluable help with in situ hybridisation and qRT-PCR, respectively, and weacknowledge Qing Zhang, Helen Prescott and Matthew Dicks for providingexcellent technical assistance. We are indebted to Miltos Tsiantis and AngelaHay for helpful discussion, and the research was funded by Oxford Universitythrough a Clarendon Scholarship to X.F., with additional financial support fromMagdalen College (Oxford).

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.049320/-/DC1

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Fig. 6. Relationships between the microsporocytes and thetapetal and middle layer cells during anther development inArabidopsis. (A,B)Following the division of the inner secondaryparietal cell (A), the cell adjacent to the microsporocyte acquires atapetal fate and is converted into a PTC (B). (C)The EMS1 complex isexpressed in the PTC cell membrane, and the MSC synthesises TPD1.(D)TPD1 activates the EMS1 complex and the PTC is induced toproliferate. Other signals induce division in the middle layer cells andthe MSCs. (E)The proliferation of the tapetal layer represses furtherMSC divisions. (F)Later the tapetal cells become fully functional,perhaps as a result of later signalling events. MSC, microsporocytes;PTC, primary tapetal cell; MLC, middle layer cell; T, functional tapetalcell.

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