RESEARCH ARTICLE 2409

Development 137, 2409-2416 (2010) doi:10.1242/dev.049320 © 2010. Published by The Company of Biologists Ltd Tapetal cell fate, lineage and proliferation in the Arabidopsis anther Xiaoqi Feng and Hugh G. Dickinson*

SUMMARY The four microsporangia of the flowering plant anther develop from archesporial cells in the L2 of the primordium. Within each microsporangium, developing microsporocytes are surrounded by concentric monolayers of tapetal, middle layer and endothecial cells. How this intricate array of tissues, each containing relatively few cells, is established in an organ possessing no formal meristems 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 in development, 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 initials fail to proliferate. Surprisingly, these cells continue to develop, isolated at the locular periphery. Mutant and wild-type microsporangia expand at similar rates and the ‘tapetal’ space at the periphery of mutant locules becomes occupied by microsporocytes. However, induction of late expression of EMS1 in the few tapetal initials in ems1 plants results in their proliferation to generate a functional tapetum, and this proliferation suppresses microsporocyte number. Our experiments also show that integrity of the tapetal monolayer is crucial for the maintenance of the polarity of divisions within it. This unexpected autonomy of the tapetal ‘lineage’ is discussed in the context of tissue development in complex plant organs, where constancy in size, shape and cell number is crucial.

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

INTRODUCTION downstream of the floral ‘C’ gene AGAMOUS (AG) (Ito et al., Unlike animals, which can initiate germlines as early as 2004) to promote sporogenous cell identity (Schiefthaler et al., embryogenesis, plant reproductive cells form late in development. 1999; Yang et al., 1999) and is reported to interact with the leucine- All male reproductive cells of Arabidopsis develop within rich repeat receptor-like kinases (LRR-RLKs) BARELY ANY concentrically organised microsporangia derived from archesporial MERISTEM 1 and 2 (BAM1 and BAM2) (Hord et al., 2006). cells in the L2 of the stamen primordium (Fig. 1A,E). These BAM1 and BAM2 are held to promote somatic cell fates, first in archesporial cells divide periclinally to form an inner primary the outward-facing products of the archesporial cell division, and sporogenous cell and an outer primary parietal cell (Fig. 1B,E). subsequently during microsporangial expansion by restricting the Primary sporogenous cells continue division to give rise to a central expression domain of SPL to the locular centre (Hord et al., 2006). sporogenous mass, and consecutive divisions of neighbouring cells In turn, SPL appears to promote BAM1 expression, possibly result in the formation of three concentric parietal layers – the creating a regulatory loop similar to that involving CLAVATA 1 tapetum adjacent to the sporogenous cells, a middle layer, and the (CLV1) and WUSCHEL (WUS) in shoot apical and floral meristems endothecium subjacent to the epidermis (Fig. 1C-E) (Canales et al., (Hord et al., 2006). 2002; Feng and Dickinson, 2007; Scott et al., 2004; Sorensen et al., Also playing an important part in microsporangial patterning, the 2002). In common with the development of other plant LRR-RLK EXCESS MICROSPOROCYTES 1 (EMS1; also reproductive organs, where consistency in size and shape is known as EXTRA SPOROGENOUS CELLS, EXS) (Canales et essential for effective function, microsporangial formation does not al., 2002; Zhao et al., 2002) is reported to form a receptor complex involve ‘classical’ plant meristems, but rather finite numbers of with two further LRR-RLKs – SOMATIC EMBRYOGENESIS divisions within apparent cell lineages – reminiscent of RECEPTOR-LIKE KINASE1 and 2 (SERK1 and SERK2) – in the development in some animal tissues. plasma membrane of tapetal cells (Albrecht et al., 2005; Colcombet Patterning of the concentric microsporangia involves the et al., 2005). This complex is held to bind TAPETUM interplay of many genes (Feng and Dickinson, 2007; Ma, 2005; DETERMINANT 1 (TPD1) (Yang et al., 2005; Yang et al., 2003), Wilson and Yang, 2004). The transcription factor a putative ligand synthesised by the microsporocyte mass, and is SPOROCYTELESS (SPL; also known as NOZZLE, NZZ) reported to specify tapetal cell fate (Albrecht et al., 2005; Feng and (Schiefthaler et al., 1999; Yang et al., 1999) acts directly Dickinson, 2007; Jia et al., 2008; Ma, 2005; Yang et al., 2005). ems1 microsporangia have no tapetal layer but excess microsporocytes (Canales et al., 2002; Zhao et al., 2002), a Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 phenotype shared with serk1 serk2 double mutant lines (Albrecht 3RB, UK. et al., 2005; Colcombet et al., 2005) and tpd1 plants (Yang et al., 2003). How this EMS1 complex mediates the formation of a *Author for correspondence ([email protected]) functional tapetal layer is unknown; models range from a role in

Accepted 14 May 2010 regulating the number of functional archesporial cells (Canales et DEVELOPMENT 2410 RESEARCH ARTICLE Development 137 (14) al., 2002) to the specification of tapetal fate in the inner division lines of Arabidopsis (Dilkes et al., 2008; Paul et al., 1992). These plants products of the inner secondary parietal cells, cells which, in ems1 form a full complement of microsporocytes and tapetal cells, but the latter mutant lines, are held to revert to a default microsporocyte fate – become highly vacuolated during the proliferation phase (S5; see Fig. S1 implying a switch in fate within a cell lineage (Zhao et al., 2002). in the supplementary material), expand rapidly and degenerate after S5, In an attempt to define further the role of the EMS1 complex in confirming pA9 to be tissue specific and active during the proliferation of the specification of the Arabidopsis tapetum and the mechanism by the tapetum. which it proliferates as a monolayer, we expressed EMS1 Light microscopy and measurements ectopically in ems1 plants after its normal period of expression. Material was prepared as previously described (Sorensen et al., 2002), These experiments show that a small population of founder cells is observed under an Olympus BX50 microscope and imaged using Image- committed to a tapetal fate very early in development, but that the Pro 6.2 software. The longitudinal mid-point of abaxial microsporangia EMS1 complex is not involved in this early specification. from the medial anthers of anther stage 6 was chosen for cell number However, the EMS1 complex is required for the subsequent measurement. Mean value and s.d. were calculated from 150 independent proliferation of these cells to form the tapetal monolayer, but not locules of each line. divisions in any other cell layer. We also show competition for Transmission electron microscopy space to exist between the products of the microsporocyte and Material was prepared as previously described (Sorensen et al., 2002). tapetal cell lineages. This early independence of the tapetal lineage and its ability to interact with neighboring cell layers provide the In situ hybridisation A 1157-bp fragment for EMS1 detection was amplified using primers first clues as to how these small but complex plant organs develop XH402F/XH402R (Zhao et al., 2002) using wild-type cDNA as template; – apparently in the absence of functional meristems. similarly, a 305-bp A9 fragment was amplified using primers XH401F/XH401R, and a fragment for A7 detection was made using DNA MATERIALS AND METHODS from vector pMC1577 kindly provided by Hong Ma (Zhao et al., 2002; Plant materials and growth conditions Rubinelli et al., 1998). Fragments were cloned into the pGEM T-Easy Wild-type (wt) Arabidopsis thaliana (Linneaus) Landsberg erecta (Ler) vector (Promega) and checked by sequencing. In situ hybridisation was plants were used unless otherwise specified. exs1, exs2, exs3 seeds were carried out on 8-mm paraffin wax-embedded sections using antisense and available in the laboratory (Canales et al., 2002). ems1 seeds were kindly sense probes for each gene as previously described (Lincoln et al., 1994). provided by Hong Ma (Pennsylvania State University, USA) (Zhao et al., 2002); tpd1 seeds by De Ye (China Agricultural University, China) (Yang Primers et al., 2003); and serk1 serk2 seeds by Sacco de Vries (Wageningen See Table S1 in the supplementary material for a list of primers used. University, Holland) (Albrecht et al., 2005). Rod Scott (Bath University, UK) provided pA9::Barnase lines in both Ler and Columbia-0 backgrounds (Dilkes et al., 2008). The ems1 line was genotyped using RESULTS XF301, XF504 and XF3R primers, and tpd1 (Yang et al., 2003) and serk1 Tapetal initials in the Arabidopsis anther are serk2 (Albrecht et al., 2005) mutants were genotyped as previously specified early, and require the EMS1 receptor described. Plants were grown at 21°C on a 16 hour light/8 hour dark cycle. kinase to proliferate RT-PCR analysis To investigate the role of the EMS1 complex in tapetal Biological replicates comprised floral buds at flower stages (Smyth et al., development, we first examined events immediately following the 1990) 1-14, collected from three plants, and three independent replicates division of the archesporial cell in wild-type Arabidopsis (Fig. 1A- were used from each line. Quantitative RT-PCR (qRT-PCR) was performed I). At this stage, and only in the region under the radius of the with an Applied Biosystems 7300 Real-Time PCR system, using anther lobe (distant from the connective), the primary parietal cell XG102F/XG102R primers for AtA9 and At/AhEF1a primers (Becher et al., and its adjacent two or three L2 cells (in transverse section) 2004) as a control. Thirty-five cycles of RT-PCR were carried out surrounding the primary sporogenous cell divide to form outer, and (PyrobestDNA polymerase, TaKaRa) using XG1206F/XG1206R primers inner, secondary parietal cells (Fig. 1C). The primary sporogenous for EMS1 and GAPDH primers (Hay et al., 2002) as a control. cells commence division at this point and, simultaneously, the inner Transgenic plants secondary partietal cells adjacent to them divide periclinally to give 4005 bp of EMS1 genomic DNA and 979 bp of the A9 promoter (pA9) rise to tapetal and middle cell layer initials, which we term primary were cloned using primers XF201/XF202, and XF203/XF204, respectively. tapetal cells (PTCs) and primary middle layer cells, respectively. The pA9::EMS1 fragment was amplified through interlaced PCR using This results in the sporogenous cells becoming surrounded by a primers XF201/XF204. This fragment was cloned into pMLBART (Gleave, small number of PTCs (Fig. 1F). Dramatic growth of the 1992) using NotI digestion. The same fragment was also cloned into microsporangium then follows, achieved principally by cell Gateway pDONR207 vector (Invitrogen) after amplification using primers expansion and anticlinal division of the investing layers, and one XL101/XL102, and finally recombined into Gateway vector pGWB16 or two further, apparently unpolarised, divisions of the sporogenous (Invitrogen). Both constructs were then introduced into ems1/+ heterozygous plants using floral dip transformation (Zhang et al., 2006). cells. The PTCs participate actively in this proliferation through The pMLBART-pA9::EMS1 and pGWB16-pA9::EMS1-6xcMYC T1 plants successive anticlinal divisions, maintaining a monolayer of cells resulting were screened for BASTA, and for Kanamycin and Hygromycin between the microsporocyte mass and the dividing middle layer resistance, respectively, and were genotyped for the transgene using (Fig. 1G). The microsporocytes then enter meiosis and produce XH201/XH203 primers and for the genomic EMS1 allele using tetrads (Fig. 1H) from which individual microspores are released XF301/XF504/XF3R primers. T4 plants from each independent transgenic (Fig. 1I) to develop, via pollen mitosis 1 and 2, into pollen grains. line were genotyped and examined for phenotype. ems1 microsporangia develop similarly to wild type prior to Determination of stage specificity of the AtA9 promoter anther stage 5 [S5, anther stages refer to Sanders et al. (Sanders et Interpretation of our experiments required precise knowledge of the timing al., 1999) (Fig. 1J)]; however, the PTCs in these lines fail to of pA9 expression in our plant lines; previous reports had only described proliferate, and as both sporogenous cells (on the inside), and the pA9 driven reporter constructs as being tapetal specific (Sorensen et al., outer layers of the microsporangium (on the outside) continue to

2002). We thus investigated tapetal ablation in pA9::Barnase expressing divide and expand, individual PTCs either remain grouped (Fig. DEVELOPMENT Tapetal development in Arabidopsis RESEARCH ARTICLE 2411

Fig. 1. Development of Arabidopsis microsporangia from L2 archesporial initials. Cross sections of anthers at different developmental stages (anther stages S2-S9). (A-D)Wild-type (WT) microsporangial pattern formation through cell divisions. (E) Cell division events. The cell lineage giving rise to reproductive cells is highlighted in pink. (F-N)Microsporangial development in WT (F-I) and ems1 mutant (J-N). ems1 and WT development is similar (F,J) until S4 when ISP cell division gives rise to PTCs and PMLCs. In WT, these proliferate and develop into tapetum and middle cell layer (G). In ems1 mutants, PTCs fail to divide and differentiate. Instead these cells (hereafter termed LPCs) remain at the periphery of the locule (red arrowheads), while MSCs continue to divide (K,M). At S6, WT MSCs enter meiosis to produce tetrads (H); these release microspores (I). Tetrads degenerate (L,N) in ems1 lines and LPCs (red arrowheads) become enlarged and vacuolate. 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, outer secondary parietal cell; PMLC, primary middle layer cell; PP, primary parietal cell; PS, primary sporogenous cell; PTC, primary tapetal cell; Sp, sporogenous cell; T, tapetum; Td, tetrad; WT, wild type. S denotes anther stages (Sanders et al., 1999). Scale bars: 20mm.

1K,L), or become separated from each other, isolated at the locular as well as solely in the LPCs of ems1 anthers (Fig. 2E-H). A7 is periphery (Fig. 1M,N). As these cells do not develop into highly expressed in the active, late tapetal cells of wild-type anthers functional tapeta in ems1 locules, we have termed them locular (see Fig. S4A in the supplementary material) and, although the peripheral cells (LPCs) from this stage onwards. On average, each loculi of ems1 plants degenerate after meiosis 2 (Canales et al., ems1 microsporangium contains 3.15±0.34 LPCs in transverse 2002), the A7 transcript can nevertheless be detected in the section at the mid-point of the abaxial microsporangium of medial hypertrophic LPCs of these anthers using in situ hybridisation (see anthers at anther stage 6. The locular content of ems1 plants Fig. S4C,D in the supplementary material). A combination of degenerates shortly following the second meiotic nuclear division position and marker gene expression thus clearly identifies the (Canales et al., 2002; Zhao et al., 2002), with LPCs showing LPCs of ems1 locules as partially developed tapetal cells. dramatic hypertrophy (Fig. 1L,N). We investigated the possibility that the LPCs in ems1 plants result from low levels of normal or aberrant EMS1 expression by The locular peripheral cells of ems1 searching for functional EMS1 transcripts in ems1 plants using RT- microsporangia express tapetal cell markers PCR (see Fig. S5 in the supplementary material). No transcripts To determine what ‘tapetal’ features are possessed by the LPCs in were detected, and further evidence that the LPCs do not result the ems1 mutants, we examined these cells using transmission from incomplete penetrance of the ems1 mutation is provided by electron microscopy (TEM) and investigated the expression of two the presence of identical numbers of morphologically similar LPCs tapetal markers, At5g07230 (A9) expressed early in development in exs-1, and exs-2 and exs-3 mutants in the C24 ecotype (data not (Paul et al., 1992) and At4g28395 (A7) expressed post meiosis shown). (Rubinelli et al., 1998). TEM analysis showed the LPCs to feature a high density of ribosomes, and active populations of Golgi bodies Ectopic expression of the EMS1 receptor kinase in and mitochondria (see Fig. S2 in the supplementary material) – all the locular peripheral cells of ems1 plants features of tapetal cells. Quantitative RT-PCR (qRT-PCR) shows stimulates their proliferation and restores tapetal A9 to be expressed in wild-type and ems1 plants – but at a lower function level than in the mutant (see Fig. S3 in the supplementary To discover whether the few LPCs present in ems1 microsporangia material). In situ hybridisation confirms the presence of A9 can be ‘reactivated’ to generate functional tapetal cells (mimicking

transcript in the developing tapetal cells of wild type (Fig. 2A-D), wild-type development), we expressed EMS1 in ems1 plants under DEVELOPMENT 2412 RESEARCH ARTICLE Development 137 (14)

Fig. 2. A9 is expressed in ems1 LPCs, and the A9 promoter drives high levels of EMS1 expression in ems1 pA9::EMS1 tapeta. (A-T)In situ hybridisations showing anthers at different developmental stages. (A-H)A9 transcript in WT (A-D) and ems1 mutant (E-H). A9 expression commences from S5: exclusively within the tapetum (C and D; green arrows) in WT, or in the LPCs (G and H; red arrows) of the ems1 mutant. (I-P)EMS1 transcript in WT (I-L) and ems1 pA9::EMS1 plants (M-P; ems1 pA9::EMS1 line #5). In WT, EMS1 is expressed in all cells of the microsporangium before S5 (I), and accumulates strongly in the tapetum (green arrows) from S5 onwards (J-L). In ems1 pA9::EMS1 plants, EMS1 is only induced in the tapetum (N-P; green arrows), but at a greater level than 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 generated by developing microspores is evident. Scale bars: 20mm.

the A9 promoter (pA9), which drives tapetal-specific expression Although pA9 is activated after EMS1 transcription in wild-type during the proliferation stage of the tapetum (see Materials and plants (Canales et al., 2002; Paul et al., 1992; Zhao et al., 2002) methods) and which, in ems1 locules, is expressed only in the (Fig. 2A-D,I-L), it must direct expression sufficiently early for the LPCs. In all 42 ems1 pA9::EMS1 and pA9::EMS1-cMYC lines transgenic tapeta to follow closely the ‘wild-type’ developmental generated, LPCs both proliferated and differentiated into apparently pathway. pA9::EMS1 rescues the ems1 mutant phenotype, but it normal tapetal cells (see Fig. 3) – to which the EMS1 protein was fails to do so in either ems1 tpd1 double or ems1 serk1 serk2 triple restricted (data not shown). All lines produced fertile pollen, mutant backgrounds, although EMS1 is strongly expressed in the despite the comparatively late expression of EMS1 when driven by LPCs of the mutant anthers (see Fig. S6 in the supplementary pA9 [EMS1 expression precedes that of A9 in the wild-type anther material). However, these transgenic plants have phenotypes very (Canales et al., 2002; Paul et al., 1992; Zhao et al., 2002)] (Fig. 2A- similar to those of ems1, tpd1 and serk1 serk2 mutants (Fig. 4), D,I-L), but each transgenic line possessed a different proportion of providing further evidence that these four genes are members of the sterile locules. The tapetum was also restored in different patterns same signalling pathway (Albrecht et al., 2005; Colcombet et al., in different lines, varying from a normal monolayer (e.g. in ems1 2005). pA9::EMS1 line #10; Fig. 3A-C) to clumps of multilayered tapetum (e.g. in ems1 pA9::EMS1 line #5; Fig. 3D-F). Inducing primary tapetal cells to proliferate in pA9::EMS1 expression in ems1 microsporangia restores tapetal transgenic plants restricts the microsporocyte function sufficiently to produce viable pollen and, because only the population LPCs express A9 in these locules, it follows that the functional As the reduction in tapetal cell number in ems1 lines is tapeta of the ‘restored’ plants must be derived from this cell type. accompanied by an increase in the microsporocyte population, we Furthermore, as the tapetum of some of the transgenic lines (e.g. investigated the relationship between microsporocyte number and ems1 pA9::EMS1 line #10) so closely resembles that of wild type, the level of tapetal proliferation in ems1 pA9::EMS1 transgenic and because the LPCs themselves resemble tapetal cells both in plants. In wild-type plants, mid-point transverse sections of position and in expression of the A9 (early) and A7 (late) tapetal medial anthers with microsporangia at stage 6 transect 7.17±0.17 markers, we conclude that the wild-type tapetal layer must also be microsporocytes, whereas similar ems1 locules contain derived from the few PTCs formed around the early sporogenous 14.38±0.32 microsporocytes. The number of microsporocytes in cells. The extra tapetal cells that are frequently generated in ems1 ems1 pA9::EMS1 microsporangia varies markedly between lines, pA9::EMS1 lines are likely to be the result of the higher activity of but they are always in excess of those in wild-type plants, and

pA9 compared with pEMS1 (Fig. 2I-P). less than those found in ems1 anthers. For example, ems1 DEVELOPMENT Tapetal development in Arabidopsis RESEARCH ARTICLE 2413

Fig. 3. pA9::EMS1 restores functional tapetum to ems1 mutant anthers. Light micrographs of sectioned anthers of ems1 pA9::EMS1 plants. Rescued tapetal cells form a continuous layer (A-C), or multiple layers (D-F) that are sometimes discontinuous (E,F). Monolayered tapeta with 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: 20mm.

pA9::EMS1 line #5 sporangia contain 10.38±0.18 microsporocytes in transverse section (see Fig. S7 in the supplementary material). Similar comparisons cannot be carried pA9::EMS1 ems1 out for tapetal cells as the number in ems1 pA9::EMS1 Fig. 4. does not restore functional tapetum to tpd1 or ems1 serk1 serk2 mutant lines. (A-L) Light micrographs of microsporangia cannot be measured accurately owing to their  sectioned anthers. (A-C)tpd1 mutant microsporangia. (D-F)ems1 tpd1 multiple division planes and irregularity in size and shape. pA9::EMS1 microsporangia. (G-I)serk1 serk2 microsporangia. (J-L)ems1 However, the fact that ems1 anthers (which contain excess serk1 serk2 pA9::EMS1 microsporangia. pA9::EMS1 rescues tapetum in microsporocytes) contain few LPCs (about three per section), ems1 but not in tpd1 or serk1 serk2 mutant backgrounds; these show transgenic ems1 pA9::EMS1 lines (which contain fewer phenotypes identical to the those of the original mutant lines (tpd1, microsporocytes than ems1 plants) have significant numbers of A-C; serk1 serk2, G-I), where PTCs (red arrows) are present either as a tapetal cells, and wild-type anthers (which possess fewer group (A,D,G,J) or individual cells (B,E,H,K). Scale bars: 20mm. microsporocytes than ems1 pA9::EMS1 lines) feature a complete tapetal monolayer, points to a clear inverse relationship between tapetal cell and microsporocyte numbers. However, even when a functional monolayer of tapetal cells is Tapetum derived from isolated locular peripheral formed in ems1 pA9::EMS1 lines (e.g. as in ems1 pA9::EMS1 line cells proliferates via periclinal and anticlinal #10), the number of microsporocytes always exceeds that of wild- divisions in transgenic plants type anthers. pA9 is activated later in anther development than Our screen of microsporangia from different transgenic lines pEMS1, and this interval between expression stages may provide an revealed a wide range of phenotypes, including plants containing opportunity for the microsporocytes to commence proliferation in the clumped or multilayered tapetum (Fig. 3D-F). We believe this, like absence of a developing tapetal layer. Significantly, in all the ems1 the higher numbers of microsporocytes (see above), to result from and pA9::EMS1 ems1 lines screened, the numbers of cells differences in the timing of EMS1 function (Fig. 5). Thus, in composing the middle, endothecial and epidermal layers were different ems1 pA9::EMS1 lines, the promoter might drive EMS1 identical to those of wild-type plants, showing that the proliferation transcription to attain an effective threshold either before or after of these cell layers is unaffected by the presence or absence of both the fragmentation of the monolayer of LPCs around the EMS1 expression and a functional tapetal layer. sporogenous cells: if before, exclusively anticlinal division takes Many ems1 pA9::EMS1 microsporangia degenerate after S9 place; if afterwards, a mixture of anti- and periclinal division (Fig. 3F), and we found a clear relationship between occurs to generate tapetal layering (Fig. 5). The association microsporocyte number, the extent of tapetal disruption and locular between the integrity of the tapetal monolayer and division polarity fertility. Lines with a large number of microsporocytes were likely is striking, and points to the polarity of division (anticlinal in this to possess more fragmented and multilayered tapetum, and a higher case) being maintained by coordinating signals from neighbouring proportion of sterile locules. For example, ems1 pA9::EMS1 line cells. Such signals might be transmitted through plasmodesmata, #5, which contains 10.38±0.18 microsporocytes in transverse or generated by mechanical stress set up in the tapetal monolayer section (the highest number among all 24 transgenic lines), has the itself. Alternatively, division polarity could be regulated by the most irregular tapetum and the largest number of sterile locules directionality of the TPD1 signal from the microsporocytes. In

(see Fig. S7 in the supplementary material). wild-type locules, this signal would be received on the inward DEVELOPMENT 2414 RESEARCH ARTICLE Development 137 (14)

Fig. 5. Model for wild-type, ems1 and ems1 pA9::EMS1 microsporangial development. Division of ISPs occurs between S3 and S4 to generate PTCs and PMLCs. In WT PTCs, the EMS1 signalling complex becomes active, stimulating proliferation. In ems1, the complex is inactive and PTCs 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 LPC layer, a tapetal monolayer is formed (as in ems1 pA9::EMS1 line #10); if afterwards, clumps of cells resulting from peri- and anticlinal division are formed (as in ems1 pA9::EMS1 line #5). The later the EMS1 functional threshold is achieved, the more time MSCs have to proliferate before inhibition by tapetal cells – either through physical competition for locular space or by signalling.

facing surfaces of the tapetal cells, whereas in ems1 pA9::EMS1 ems1 plants, and that ectopically expressed EMS1 stimulates lines, where LPCs are isolated, the TPD1 signal could be perceived proliferation of only these cells in the latter indicate that an on their radial (and perhaps outward) surfaces. Such a system has alternative interpretation of these events might be required. been reported for stomatal development, where the LRR-RLKs ERECTA (ER), ERECTA LIKE 1 (ERL1) (Shpak et al., 2005) and The specification, proliferation and maintenance TOO MANY MOUTHS (TMM) (Geisler et al., 2000; Nadeau and of the Arabidopsis tapetal cell lineage Sack, 2002) form a heterodimer, which orients the cell division The new data point to the small number of cells formed adjacent plate depending upon from which direction it receives the ligand to the sporogenous cells immediately becoming committed to a EPIDERMAL PATTERNING FACTOR 1 (EPF1) (De Smet et al., ‘pre-tapetal’ fate, and the EMS1 signalling pathway being 2009; Hara et al., 2007). responsible for the subsequent proliferation of these cells, allowing the tapetal monolayer to keep pace with the rapid expansion of the DISCUSSION other layers of the loculus, which, because they are unaffected by The sequence of cell divisions leading to tapetal formation is well the absence of functional EMS1, must be regulated by different understood in Arabidopsis (Canales et al., 2002; Feng and signals. This role for EMS1 in proliferation implies that the EMS1 Dickinson, 2007; Scott et al., 2004), but how cells generated by the complex cannot be involved in the initial establishment of tapetal periclinal division of the inner secondary parietal (ISP) cell layer fate in the PTCs; the genes responsible for this are unknown, but acquire and maintain their tapetal fate is unclear. The similarity in other LRR-RLKs, such as BAM1/2, remain possible candidates. cell numbers between wild type and ems1 mutants (where no Arguably, the LPCs of ems1 anthers could represent a small tapetum is formed) has reasonably been interpreted as evidence that number of partially developed tapetal cells resulting from the these ISP cells revert to a default microsporocyte fate in the incomplete penetration of the ems1 mutation, with the remainder absence of the EMS1 receptor kinase (Zhao et al., 2002). However, of the inner division products of the inner secondary parietal cell our findings that a small number of cells with tapetal features being converted into microsporocytes. This is unlikely because: (1)

(PTCs/LPCs) are formed early in development in wild-type and EMS1 transcripts are absent from ems1 mutant plants; (2) DEVELOPMENT Tapetal development in Arabidopsis RESEARCH ARTICLE 2415

of the developing monolayer requires its continued integrity, revealing that anticlinal divisions within it are either controlled by internal signals, or by microsporocyte-generated TPD1 acting as a directional cue. Cells of the tapetum and the microsporocytes thus develop as ‘communicating’ neighbours, but as independent lineages occupying defined domains within the expanding microsporangium. Whether the proliferation of microsporocytes into ‘tapetal territory’ in ems1 lines results from the lack of signals from the tapetum, or simply from available space, remains to be determined. Fig. 6. Relationships between the microsporocytes and the Perhaps the most significant difference between development of tapetal and middle layer cells during anther development in these complex microsporangial tissues and development of those Arabidopsis. (A,B)Following the division of the inner secondary derived from the apical and root meristems is their early and parietal cell (A), the cell adjacent to the microsporocyte acquires a continued independence; certainly they can receive both directional tapetal fate and is converted into a PTC (B). (C)The EMS1 complex is and ‘checkpoint’ signals and perhaps alter the space they occupy expressed in the PTC cell membrane, and the MSC synthesises TPD1. but, once formed, their founder cells generate independently (D)TPD1 activates the EMS1 complex and the PTC is induced to proliferating lineages committed to single developmental fates. proliferate. Other signals induce division in the middle layer cells and Without this degree of independence and level of ‘internal’ control the MSCs. (E) The proliferation of the tapetal layer represses further  it is difficult to see how small numbers of cells of different fates MSC divisions. (F)Later the tapetal cells become fully functional, perhaps as a result of later signalling events. MSC, microsporocytes; can assemble into such a range of intricate patterns within a small, PTC, primary tapetal cell; MLC, middle layer cell; T, functional tapetal terminally differentiated organ such as the anther. cell. Acknowledgements We thank the following for providing mutant lines and reagents: Hong Ma, De Ye, Sacco De Vries, and Rod Scott for providing the pA9::Barnase lines and reactivation of these few LPCs in ems1 lines can generate a information on A9 expression patterns. Carla Galinha and Paolo Piazza gave complete and functional tapetum that supports the production of valuable help with in situ hybridisation and qRT-PCR, respectively, and we viable pollen; and (3) LPCs are present in all plants carrying ems1 acknowledge Qing Zhang, Helen Prescott and Matthew Dicks for providing excellent technical assistance. We are indebted to Miltos Tsiantis and Angela alleles in both Ler and C24 backgrounds (some encoding non- Hay for helpful discussion, and the research was funded by Oxford University functional receptor kinases), as well as in tpd1 and serk1 serk2 through a Clarendon Scholarship to X.F., with additional financial support from plants. Magdalen College (Oxford). Although the EMS1 complex is required for the proliferation of PTCs, its role in their final differentiation into functional tapetal Competing interests statement The authors declare no competing financial interests. cells is unclear. LPCs certainly complete some aspects of tapetal development in ems1 lines as they express A7, a gene encoding a Supplementary material lipid transfer protein that is normally expressed post meiosis Supplementary material for this article is available at (Rubinelli et al., 1998), but they nevertheless fail to develop into http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.049320/-/DC1 functional tapetal cells. The EMS1 complex and its putative ligand References TPD1 thus emerge as a component of a larger gene network Albrecht, C., Russinova, E., Hecht, V., Baaijens, E. and de Vries, S. (2005). The regulating tapetal development, with other network members being Arabidopsis thaliana somatic embryogenesis receptor-like kinases 1 and 2 control male sporogenesis. Plant Cell 17, 3337-3349. responsible for specifying initial tapetal fate in the PTCs and, Becher, M., Talke, I. N., Krall, L. and Kramer, U. (2004). Cross-species perhaps, aspects of subsequent development. 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