patterning during ovule development

INTRODUCTION

The ovule of Arabidopsis thalianahas become a prominentmodel system for the genetic and molecular study of floralorganogenesis (Gasser et al., 1998; Schneitz, 1999; Schneitz etal., 1998b). It is located within the gynoecium, often normallycomposed of two carpels, and represents the progenitor of theseed. As such the ovule is the major female reproductive organin higher plants. Within the ovule the egg cell is formed,fertilization occurs and subsequently, during the ontogeny ofthe seed, the embryo develops.

Pattern formation represents an important aspect of earlyovule development. For example, several distinct regions canbe identified morphologically along a proximal-distal (PD),or chalazal-micropylar, axis. Distally, the nucellus ischaracterized by the presence of the megaspore mother cell andeventually the embryo sac, which harbors the egg cell. Thenucellus thus corresponds to the megasporangium. Centrally,usually two integuments that initiate at its flanks identify thechalaza. Proximally, the stalk or funiculus is characterized bythe development of the vascular strand. This PD arrangementof different regional identities is recognizable at a very earlystage in Arabidopsis thalianaindicating that it becomesestablished during the early proliferative phase of ovuleprimordium formation (Schneitz et al., 1995). Furthermore, itis highly conserved during evolution and thus typical for ageneralized seed plant (Esau, 1977).

How is this pattern set up? We have proposed that an earlycorresponding prepattern is established during early ovuledevelopment (Schneitz et al., 1995). The distal pattern elementor domain is the progenitor of the nucellus, the central patternelement develops into the chalaza and the proximal domainoriginates the funiculus. Part of the genetic evidence is basedon the observation that many mutations that cause early defectsin ovule development lead to corresponding region-specificdefects within the ovule, leaving early development in otherregions largely intact (Gasser et al., 1998; Schneitz, 1999;Schneitz et al., 1998b). In addition, it was shown that the distalelement is established in an independent fashion because anucellus develops even in the absence of the subnucellar region(Schneitz et al., 1998a). Early molecular support has comefrom studies on the BELL(BEL1) gene (Reiser et al., 1995).The BEL1expression pattern provides evidence for the centralpattern element as its expression within the ovule primordiumgets restricted to a central band, approximately correspondingto the central domain, prior to morphological signs ofintegument differentiation.

Intriguing questions arise from such considerations. Forexample, what genes are involved in primordium outgrowthand PD pattern formation and how do they interact toorchestrate these aspects of ovule development? Known genesthat control primordium emergence include AINTEGUMENTA(ANT) and HUELLENLOS(HLL). In ant mutants only abouthalf the regular number of primordia are present and the ovules

4227Development 127, 4227-4238 (2000)Printed in Great Britain © The Company of Biologists Limited 2000DEV0322

With the characterisation of the NOZZLEgene we aim ata better understanding of the molecular and geneticmechanism underlying pattern formation and growthcontrol during floral organogenesis. Our data indicate thatNOZZLE links these processes during ovule development.In the ovule primordium NOZZLE plays a central role inthe formation of the nucellus through antagonizing theactivities of BELL, AINTEGUMENTA and INNER NOOUTER, all encoding putative transcription factors, in theprospective nucellar region. We provide evidence thatNOZZLE and BELL are chalaza identity genes that shareoverlapping functions in establishing the prospectivechalaza of the ovule. In addition, NOZZLE plays a role in

controlling the cell number and by this means thelength of the funiculus, again through antagonizingAINTEGUMENTA and INNER NO OUTER function.NOZZLE is also required for the development of theinteguments. We show that during the initial phase ofthis process NOZZLE is transcriptionally regulated byAINTEGUMENTA and INNER NO OUTER. NOZZLE thusrepresents a downstream target of these two genes in theintegument development pathway.

Key words: Arabidopsis thaliana, Pattern formation, Ovule,NOZZLE, Organogenesis, Sporogenesis

SUMMARY

NOZZLE regulates proximal-distal pattern formation, cell proliferation and

early sporogenesis during ovule development in Arabidopsis thaliana

Sureshkumar Balasubramanian and Kay Schneitz*

Institute of Plant Biology, University of Zürich, Zollikerstr. 107, CH-8008 Zürich, Switzerland*Author for correspondence (e-mail: [email protected])

Accepted 25 July; published on WWW 7 September 2000

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are slightly shortened. In addition, integument developmentfails to proceed beyond the initial steps, indicating that ANTplays a role in integument development as well (Baker et al.,1997; Elliott et al., 1996; Klucher et al., 1996; Schneitz et al.,1998a; Schneitz et al., 1997). ANT regulates growth and cellproliferation in several types of plant organs (Baker et al.,1997; Elliott et al., 1996; Klucher et al., 1996; Krizek, 1999;Mizukami and Fischer, 2000) and it has been postulated thatANT maintains the meristematic competence of cells duringorganogenesis (Mizukami and Fischer, 2000). The ANT locusencodes a putative transcription factor of the AP2 class (Elliottet al., 1996; Klucher et al., 1996). Shorter ovules and missinginteguments are also observed in hllmutants and HLLand ANTshare redundant functions in ovule primordium outgrowth(Schneitz et al., 1998a). Mutations in INNER NO OUTER(INO) do not lead to an obvious defect in primordiumoutgrowth; however, they do cause an early block in outerintegument development (Gaiser et al., 1995; Schneitz et al.,1997; Villanueva et al., 1999). Interestingly, INO encodes aputative transcription factor of the YABBY class (Villanuevaet al., 1999) and members of this group of proteins are involvedin mediating abaxial cell specification in Arabidopsis(Bowman, 2000; Bowman and Smyth, 1999; Chen et al., 1999;Sawa et al., 1999; Siegfried et al., 1999).

Very few known loci have been implicated to function in PDpattern formation. In fact, the BEL1gene has been the onlygood candidate for quite some time. BEL1 encodes ahomeodomain putative transcription factor (Reiser et al.,1995). In bel1mutants the chalazal domain undergoes altereddevelopment and irregular outgrowths of unknown identityform instead of integuments (Modrusan et al., 1994; Robinson-Beers et al., 1992; Schneitz et al., 1997). The biologicalfunction of BEL1remains enigmatic as the data are compatiblewith both a direct role of BEL1 in establishing the chalazalidentity, and with a role in chalaza-specific growth control inresponse to PD pattern formation (Reiser et al., 1995). Morerecently the NOZZLE(NZZ) gene has been considered as asecond prominent candidate (Schiefthaler et al., 1999). Apreliminary analysis suggested an early role for NZZ insporangium formation and sporogenesis. In nzz mutants thenucellus and the pollen sacs are strongly reduced and thefounder cells of the sporogeneous lineage fail to develop. TheNZZ locus encodes a putative protein of unknown biochemicalfunction. However, computer-based sequence analysis raisedthe possibility that NZZ represents a nuclear protein andpossibly a transcription factor (Schiefthaler et al., 1999).Recently, a mutant termed sporocyteless(spl) was identifiedand found to be impaired in sporocyte differentiation (Yang etal., 1999). Sequence analysis of the SPLgene showed it to beidentical to NZZand further experiments indicated that the SPLprotein is localised in the nucleus.

Here we present a detailed characterisation of NZZlargelyfocusing on its function in ovule development.

MATERIALS AND METHODS

Plant work and geneticsPlants were grown as described by Schneitz et al. (1997) andArabidopsis thaliana(L.) Heynh. var. Landsberg (erectamutant) wasused as the wild-type strain. The isolation and molecular

characterization of the mutant nzzalleles has also been described(Schiefthaler et al., 1999).

For the double-mutant analysis the following alleles were used: nzz-2, nzz-1, bel1-1460 and ant-72F5 (Schneitz et al., 1997), ino-2(Schneitz et al., 1997; Villanueva et al., 1999) (previously known asino-46E4), hll-2 (Schneitz et al., 1998a), bel1-3(Modrusan et al.,1994; Reiser et al., 1995) and aberrant testa shape(ats; Léon-Kloosterziel et al., 1994). Double mutants were identified by theirnovel phenotypes segregating in the expected frequencies and becausethey exhibit a nzz-like anther phenotype in addition to the ovulephenotype. Between 14 and 20 individual double mutants wereobtained, depending on the combination, except for nzz-2 ant-72F5(6/1573; double mutants/scored plants) double mutants. The ANTgene (Elliott et al., 1996; Klucher et al., 1996) maps closely to NZZrendering it difficult to isolate the corresponding double mutantplants.

In situ hybridization, ovule preparations, microscopy andart workIn situ hybridization experiments, including the generation of NZZ,BEL1 and ANT antisense transcripts, were carried out as describedpreviously (Schiefthaler et al., 1999; Schneitz et al., 1998a). In situhybridization using the INOprobe was performed using a differentprotocol (Vielle-Calzada et al., 1999) with minor modifications. Nearfull-length INO antisense and sense transcripts were generated fromplasmid pJMV50 (Villanueva et al., 1999) after linearising thetemplate with PstI and XhoI and performing the in vitro transcriptionreaction with T7 and T3 RNA polymerase, respectively. This antisenseprobe yielded identical results compared to previously obtained datausing a probe lacking the information for the YABBY domain(pJMV86; Villanueva et al., 1999). Light microscopy, scanningelectron microscopy, staging, whole-mount ovule preparations andartwork have also been described earlier (Schiefthaler et al., 1999;Schneitz et al., 1998a, 1997, 1995).

RESULTS

Wild-type ovule developmentWild-type ovule development in Arabidopsishas been welldescribed previously (Christensen et al., 1997; Mansfield et al.,1991; Modrusan et al., 1994; Robinson-Beers et al., 1992;Schneitz et al., 1995; Webb and Gunning, 1990; Fig. 1A-D,I-L). During stage 1 the primordium forms (stages according toSchneitz et al., 1995). During stage 2 the megaspore mothercell differentiates within the nucellus and enters meiosisfollowed by cytokinesis. Concomitantly to meiosis the twointeguments initiate from the chalaza, each originallyconsisting of two cell layers, and begin to grow around thenucellus in an asymmetric fashion. This uneven growth willeventually lead to the characteristic kinked (anatropous) shapeof the mature ovule. During stage 3 the functional megasporedevelops into the embryo sac, integument ontogenesis proceedsand the vascular strand becomes visible within the funiculus.At the end of stage 3 prefertilization development has endedand the mature ovule is ready for postfertilization developmentwhich begins with stage 4. For the most part the ovule consistsof L1 and L2-derived tissue (Jenik and Irish, 2000).

The ovule aspect of the nzz-2 mutant phenotypeWe have isolated three mutant alleles of NZZ (Schiefthaler etal., 1999). All three mutant nzz alleles behave as recessivehypomorphs and exhibit a Mendelian segregation pattern (not

S. Balasubramanian and K. Schneitz

4229Patterning during ovule development

shown). The major defects appear to be restricted to ovule andanther development, thus the mutant plants exhibit male andfemale sterility (Schiefthaler et al., 1999) (Fig. 1). Theinflorescence often appears more compact as well. Thephenotypes of the three mutants are quite similar, however, thealterations in nzz-2are somewhat stronger than the defects innzz-1and nzz-3. Therefore, we focus on a description of theovule aspects of the nzz-2phenotype. The nzz-2mutant carriesa point mutation in exon 1 of the NZZgene resulting in a stopcodon. The putative wild-type protein has a length of 314residues. In contrast the mutant protein is shortened to 142residues. This shorter protein lacks a putative nuclearlocalisation signal and the D1/D2 region (Schiefthaler et al.,1999).

An obvious defect in nzz-2mutants is the apparent absenceof the prominent megaspore mother cell (MMC) at stage 2-I(Fig. 1M) and a strongly diminished nucellar tissue, which isbest seen at stages 2-II/III (Fig. 1F,N). At these stages anucellus is readily apparent in wild type because of thepresence of the MMC (Fig. 1I), and because the nucellus forms

a distinct dome atop the developing integuments (Fig. 1B). Wehave scored ovules of wild type and nzz-2mutants for thepresence of MMCs (at stage 2-III) and embryo sacs (at stages4-V) (Table 1). All ovules from wild-type plants showed aMMC or embryo sac. In nzz-2mutants only 5% of ovules atstage 2-III showed a MMC in a still somewhat reducednucellus (Table 1). Because we cannot detect a MMC it is notclear whether the much reduced distal part in nzz-2mutantsactually represents a nucellus, and thus we refer to it as thedistal tip region. The development of the nucellus is not simplydelayed in nzz-2mutants, because the ovule continuesdevelopment, and, while in about 10% of cases a tiny distal tipregion can be seen, a regular-size nucellus is usually notobserved even at stage 4-V (Fig. 1P; Table 1). In this contextit is noteworthy that two large groups of mutants with a defectin megasporogenesis and/or embryo sac development exist andthat in those mutants a nucellus can be observed (Schneitz etal., 1997). We have quantified the reduction by counting theepidermal cells in the distal part of the primordium in a mid-sagittal plane of a single profile flanked by the inner integument

Fig. 1.Ovule developmentin wild type and in nzz-2mutants. (A-H) Scanningelectron micrographs(SEMs). (I-P) Verticaloptical sections throughwhole-mount ovules.(A-D,I-L) Wild type.(E-H,M-P) The nzz-2mutant. Stages: (A,E,I,M) 2-I; (B,F) early 2-III, (J,N) late2-III; (C,G,K,O) 3-IV;(D,H,P) about 4-V; (L) late3-VI. (B) The nucellus isprominent in wild type.(F) Note the reduced extentof the distal tip region(arrow) and compare with B.(G) The arrow highlights arare instance where thenucellus and integumentsare absent and just afuniculus is seen. (K) Thearrow points to a four-nuclear embryo sac. Onlytwo nuclei can be seen inthis optical section.(N) Inner integumentdevelopment is delayed. Thearrow denotes the regionwhere the inner integumentwill develop. (O) A nucellusor embryo sac cannot bedetected (compare with K).(P) The prominent anteriorand posterior endotheliumcan bee seen to be in closeassociation. No nucellus orembryo sac is discernible(arrow) as in L. cc, centralcell; ec, egg cell; et,endothelium; fu, funiculus, ii, inner integument; mmc, megaspore mother cell; mp, micropyle; nu, nucellus; oi, outer integument; pt, pollentube; syn, synergid; vs, vascular strand. Scale bars, 20 µm.

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at stage 2-IV/V. 17.6±2.2 (mean±s.d.) cells were present innucelli of wild-type plants (n=17), in contrast to 7.7±2.1 cellsin the distal tip region of nzz-2mutants (n=20). Thus, whilebeing somewhat variable (Fig. 1F) the extension of the distaltip region is routinely only about 40% compared to the nucellarextension in wild type. The entire PD extension of stage 1-II/2-III primordia does not appear to be notably reduced in nzz-2mutants when compared to wild type (Fig. 1A,B,E,F). As theMMC is usually not detected, the defect is likely to occur priorto or around stage 2-I (see also below). However, we cannotexclude the possibility that nzz-2mutants are defective innucellar growth during early to mid-stage 2.

In addition to the defect in the formation of the nucellus thetemporal arrangement of integument initiation is altered in nzz-2mutants. In wild type, the inner integument often initiates slightlyearlier (stage 2-II) than the outer integument (stage 2-III)(Robinson-Beers et al., 1992; Schneitz et al., 1995) (Fig. 1B). Innzz-2mutants the outer integument is usually seen first (Fig.1F,N). While regular integuments may develop (Fig. 1P) bothinteguments often appear reduced at later stages (Fig. 1G,O). Inrare instances the integuments are even absent, resulting in ovulesconsisting only of a funiculus (Fig. 1G). Furthermore, the lengthof the funiculus is also affected. The funiculus is extended dueto a larger number of cells (Fig. 1F-H; Table 1). This isparticularly visible during mid to late ovule development.

In summary, we find that in nzz-2mutants the distal regionis strongly reduced or missing, a prominent MMC is usuallyabsent, the integuments can be variably shortened, and thefunicular PD extension increases due to extra cell proliferation.

Double mutant analysisIn order to investigate the genetic interactions of NZZand other

known genes with a function during early ovule developmentwe analysed a series of corresponding double mutants (seeMaterials and Methods).

nzz ats and nzz hllABERRANT TESTA SHAPE(ATS) is important for integumentdevelopment since in the atsmutant integument development


Table 1. Nucellar and funicular development in wild type and various mutantsPresence of MMC (st. 2-III) Presence of nu/dt (st. 4-V) Length of fu (st. 4-V)

No. of ovules No. of ovules No. of ovulesGenotype with MMC Total % with nu/dt Total % No. of cells*,‡ counted %

Ler 70 70 100 267 267 100 21/67±1/44 15 100nnz-2 15 334 5 21 188 11 31.16±2.88 25 144nzz-2 ino-46E4 78 78 100 278 278 100 20.25±1.81 20 100ant-72F5 87 87 100 97 97 100 13.46±2.00 15 62nzz-2 ant-72F5 37 61 60 186 186 100 15.55±2.18 20 72bel1-1460 83 83 100 129 129 100 14.43±1.87 16 66nzz-2 bel1-1460 8 149 5 138 138 100 36.00±2.81 21 166nzz-2 bel1-3 ND ND 41.40±3.94 20 191

*Epidermal cells within a central-adaxial file extending from the micropylar proximal edge of the outer integument to the placenta. In the case of nzz-2 bel1-1460 and nzz-2 bel1-3 all epidermal-looking cells were included. In ino-2 the position of the proximal edge of the outer integument-like protrusion, and in bel1-1460 the proximal edge of the central region outgrowths were used as distal boundary.

‡Mean ± s.d.Abbreviations: nu, nucellus; dt, distal tip region; fu, funiculus; MMC, megaspore mother cell.

Fig. 2.SEMs of the nzz-2 atsand nzz-2 hll-2double mutantphenotypes. (A,C,E,G) Stage 2-III, (B,D,F,H) about 4-V stage.(A,B) ats, (C,D) nzz-2 ats, (E,F) hll-2, (G,H) nzz-2 hll-2. (A) The twointeguments are difficult to discriminate (arrow). Compare with Fig.1B. (C) The distal tissue size is reduced. The delay of innerintegument initiation still occurs even though it is less obvious.However, an outer integument is recognizable. (D) Note the lack ofcurvature of the micropylar half of the outer integument. (F,H) Thearrows indicate the occurrence of cell death resulting in collapsedtissue. dt, distal tip region; nu, nucellus; oi, outer integument. Scalebars, 20 µm.


is impaired leading to a single ‘fused’ integument (Léon-Kloosterziel et al., 1994; Fig. 2A,B). Except for the failure ofthe ovule to reach an anatropous shape (resembling ovulesfrom superman(sup) mutants; Gaiser et al., 1995; Schneitz etal., 1997) the nzz atsdouble mutants exhibit a largely additivephenotype (Fig. 2C,D). HUELLENLOS(HLL) is repeatedlyrequired for growth control during ovule development(Schneitz et al., 1998a, 1997). In hll mutants the ovule

primordia are shorter, the integuments fail to develop beyondthe initial steps and many cells die (Fig. 2E,F). The nzz hlldouble mutants exhibit an additive phenotype as well (Fig.2G,H). The results indicate that NZZ functions in a differentpathway than ATSor HLL during ovule development.

nzz bel1Ovules of bel1mutants lack the inner and outer integument

Fig. 3.SEMs of ovule developmentin different single and doublemutants. (A-D) bel1-1460.(E-H) nzz-2 bel1-1460. (I-L) ino-2.(M-P) nzz-2 ino-2. (Q-T) ant-72F5.(U-X) nzz-2 ant-72F5. Stages:(A,E,M) about 1-II/2-I;(I,Q,U) about 1-I/1-II;(B,F,J,N,R,V) about 2-III/IV;(C,G,K,O,S,W) about 3-IV;(D,H,L,P,T,X) about 4-V.(F,G) Neither integuments nor bel1-like outgrowths are detectable.(H) Compare the epidermal cellmorphology (arrow) with thefunicular epidermal cellmorphology in Fig. 1D. Note thebifurcated tips of the structures.(K) Note the adaxial cellenlargements (arrow).(N,O) Compare the size of thedistal tip region with the size of thenucellus in (J,K). Abbreviations: dt,distal tip region; ii; innerintegument; nu, nucellus; oi, outerintegument. Scale bars, 20 µm.

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and exhibit multicellular protuberances which emerge fromthe central region (Modrusan et al., 1994; Robinson-Beers etal., 1992; Schneitz et al., 1997). The nzz-2 bel1-1460doublemutants show a synergistic phenotype that is restricted tothe ovules (Figs 3E-H, 4AC,D). Such plants originateconspicuous elongated structures instead of true ovules. Asjudged by the morphology of the epidermal cells the majorityof such a structure resembles, an albeit much too long,funiculus. In nzz-2 bel1-3double mutants this effect is morepronounced than in nzz-2 bel1-1460mutants (Table 1).However, with a few exceptions, a vascular strand is notroutinely detected (data not shown). The distal tip regionconsists of small uniform cells and is often found bi-ortrifurcated at late stages. It is difficult to assess the extent ofthe distal tissue at early stages. MMCs were observed in thistip region at a frequency comparable to that of nzz-2singlemutants (Table 1). Similar results were obtained with nzz-1bel-1460and nzz-2 bel1-3double mutants (data not shownand Table 1). This indicates that NZZ and BEL1are stillactive in bel1and nzzmutants, respectively, and that the twogenes have overlapping functions in the specification of thechalaza.

nzz inoINNER NO OUTER(INO) has a prominent role in outerintegument development (Baker et al., 1997; Schneitz et al.,1997; Villanueva et al., 1999). In ino mutants the outerintegument development does not proceed beyond theinitial epidermal cell enlargement (Figs 3I-L, 4E,F).Megasporogenesis takes place but embryo sac developmentceases around stage 3-IV or later.

In nzz-2 ino-2double mutants (Figs 3M-P, 4G,H), and incontrast to nzz-2mutants, the distal region appears to bepresent at full size. However, only 20% of young ovules atstage 2-III show a MMC (Table 1). At later stages the distaltissue continues to grow and often protrudes from a micropylewhich is formed by a somewhat shorter inner integument. Theouter integument fails to develop as in ino mutants and thefuniculus is of about normal length (Table 1). Comparableresults were obtained with nzz-1 ino-2double mutants (data notshown).


Fig. 4. Optical sections through whole-mount ovules of differentsingle and double mutants. The same mutants as in Fig. 3. Stages:(A,C,E,G,K) 2-III; (I) 2-V, (B,D,F,H,J,L) about 4-V. (A,B)bel1-1460. (C,D)nzz-2 bel1-1460. (E,F)ino-2. (G,H) nzz-2 ino-2.(I,J) ant-72F5. (K,L) nzz-2 ant-72F5. (A) A megaspore mother celland the bulging of the developing outgrowths can be seen at a youngstage. (C) The arrow highlights the absence of a MMC. (D) NoMMC can be seen in the tri-furcated distal tip region (arrow).(E) Note the presence of a MMC (arrow). Inner but not outerintegument initiation can be seen. (F) A differentiated innerintegument is present as judged by the presence of the endothelium.(G) Note absence of MMC in distal tip (arrow). (H) An innerintegument is visible, however, an endothelium is not alwaysapparent. The arrow denotes the protruding mass of cells that isdevoid of a MMC or embryo sac. (I) A tetrad is regularly seen.(K) An ovule with a MMC. (L) A nucellus is prominent and noembryo sac is apparent and the outer integument is partiallydeveloped. et, endothelium; ii, inner integument; mmc, megasporemother cell; oi, outer integument; tet, tetrad; vs, vascular strand.Scale bars, 20 µm.


It appears that loss of INO function partially restoresnucellar development and suppresses the extra growth of thefuniculus in the absence of normal NZZ function.

nzz antBesides other functions, AINTEGUMENTA(ANT) has animportant role during ovule primordium outgrowth andintegument development (Baker et al., 1997; Elliott et al.,1996; Klucher et al., 1996; Schneitz et al., 1998a, 1997). In ant

mutants sporogenesis occurs (Fig. 4I; Table 1) but subsequentembryo sac development is aberrant. In addition, theintegument development is blocked at an early stage and rarelyproceeds beyond the epidermal cell enlargement of the outerintegument (Figs 3R-T, 4I,J). Finally, the funiculus is reduced(Schneitz et al., 1998a; Table 1).

Similarly to nzz inodouble mutants, nucellar developmentappears to be partially restored in plants simultaneouslydefective for NZZand ANTfunction (Figs 3U-X, 4K,L). At

Fig. 5.The NZZexpression pattern in young ovules from wild type, bel1, antand inomutants detected by in situ hybridization. Longitudinaltissue sections are shown except for M (horizontal section). Stages: (I,M) 1-I; (A,E) 1-II; (J,N) 2-I; (B,F) 2-II/III; (C,K,O) 2-III; (D,G,L,P) 2-IV; (H) 3-I. (A-D) wild type; (E-H) bel1-1460; (I-L) ant-72F5; (M-P) ino-2. (B) Note the strong signal in the developing integuments (arrows).(G,H) Strong NZZexpression can be detected in the developing outgrowths of the ovules of bel1mutants (arrow). (K,L) Strong epidermalstaining of NZZin the chalazal region cannot be detected in antmutants (arrows). (O,P) Note the lack of strong epidermal staining in the regionnext to the inner integument where the outer integument normally develops (arrows). Abbreviations: fu, funiculus; ii, inner integument; mmc,megaspore mother cell; oi, outer integument. Scale bars, 20 µm.

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stage 2-III the distal tip region seems fully grown. A MMC isobserved in 60% of these mutants (Table 1) but it neverdevelops into an embryo sac. At late stages the outerintegument is slightly more advanced than in ant mutants,

while the funiculus appears to be of similar size to that of antsingle mutants (Figs 3X, 4L; Table 1).

These data indicate that the establishment of the nzzmutantphenotype requires ANTfunction as well.



In situ hybridization studiesWe tested whether the observed interactions between NZZand BEL1, INO and ANT are reflected at the transcriptionallevel with a set of in situ hybridization experiments (Figs 5,6).

The expression of NZZ is altered in ovules of bel-1 ,ant and ino mutantsIn wild type, strong expression of NZZ transcripts can bedetected throughout the ovule primordium at stage 1(Schiefthaler et al., 1999; Fig. 5A-D). During stage 2 NZZexpression is still detected everywhere but at a lower level.Stronger epidermal expression can be seen in the chalaza. Itstarts prior to visible signs of integument initiation andcontinues to be present in the developing integuments (Fig. 5B-D). We first tested NZZexpression in nzz-2mutants and foundno deviation from the normal pattern (not shown). In bel1mutants early expression of NZZappears normal (Fig. 5E,F) asis the weaker overall expression at slightly later stages.However, strong staining is detected in the developing irregularoutgrowths (Fig. 5G,H).

The overall early strong and later weaker expression of NZZis also observed in ovules of ant and inomutants (Fig. 5I-P).Often it appears as if the staining in the MMC is somewhatmore pronounced in ant and ino(and bel1) mutants comparedto wild type. In ant mutants the generally weaker staining isalso observed in the chalazal epidermis at stages 2-II/III whenin wild type the initiating integuments exhibit the stronglabeling (compare Fig. 5J,K with 5B,C). In ino mutants, whileNZZ is strongly expressed in the developing inner integument,only weak labeling is detected in the chalazal epidermis at theposition where the outer integument normally develops (Fig.5N-P). These results indicate that ANTand INOact as director indirect upstream transcriptional regulators of NZZduringearly integument development.

The expression pattern of BEL1, ANT and INO inovules of nzz-2 mutantsThe BEL1pattern appears normal at early stage 1 (Fig. 6A,E).Around late stage 1-II in wild type, BEL1 expression is

excluded from the distal and proximal regions and thus thepattern resembles a central stripe approximately marking theprospective chalaza (Reiser et al., 1995; Fig. 6B,C). Slightlylater, particularly the outgrowing integuments exhibit strongBEL1expression but the rest of the chalaza and a distal part ofthe funiculus stain as well (Fig. 6D). In nzz-2mutants a centralstripe is generally not observed and the BEL1pattern occupiesthe distal-half of the primordium (Fig. 6F,Gb,Hb). While thepattern is clearly widened distally it is likely to be broadenedproximally as well since at around stage 2-IV/V a considerablepart of the funiculus, compared to wild type, is labeled by theBEL1probe (Fig. 6Hb). In some cases (less than 5%) a stripecan be detected (Fig. 6Ga,Ha). Those examples probablycorrespond to the few specimens that regularly develop moredistal tissue and eventually a MMC (compare Table 1). Theresult indicates that in nzz-2mutants BEL1 mRNA isectopically expressed in the distal region of the ovuleprimordium around late stage 1/early stage 2.

In wild type the ANTgene is expressed ubiquitously in theovule primordium at early stage 1 (Elliott et al., 1996; Fig.6I). By late stage 1/early stage 2 ANTexpression can be foundin a central domain (Fig. 6Ja,Jb) and during later stage 2particularly in the developing integuments and in most of thedeveloping funiculus (Elliott et al., 1996; Fig. 6Jb,K,L). Innzz-2mutants ANT expression, during early stage 1, is againnormal. At late stage 1/early stage 2 the ANTand BEL1patterns are in part comparable since ANT RNA is alsodetected in the distal-half of the primordium (Fig. 6N,O).However, some stronger staining is regularly detected in afew cells of the epidermis at an abaxial (posterior) location(arrows). The cells at this position are likely to be part of thedeveloping outer integument which initiates in a similarlyasymmetric fashion at the abaxial side of the primordium(Robinson-Beers et al., 1992) and which becomes visibleprior to the inner integument in nzzmutants (Fig. 1F,N).Later, ANT expression is found throughout both developinginteguments and most of the funiculus (Fig. 6P). As with theBEL1probe, in a few specimens a less-stained region can beseen at the distal tip (Fig. Pa). In summary, we observeectopic expression of ANTmRNA in the distal region of theovule primordium of nzz-2mutants around late stage 1/earlystage 2.

Fig. 6. The BEL1, ANTand INOexpression patterns in young ovulesfrom wild type and nzz-2mutants detected by in situ hybridization.Longitudinal sections through ovules are shown except for A,E,I andQ, which are horizontal sections through carpels, and T, whichfeatures a tangential section at a distal-to-central position. Stages:(A,E,I,M) 1-I; (B,F,Ja,N,Q) 1-II; (C,Ga,Gb,Jb,R,U) 2-I; (O) 2-II;(K,V) 2-III; (D,Ha,L,S,T,W) 2-IV; (Hb,Pa,Pb,X) 2-V/3-I. Expressionpatterns: (A-H)BEL1; (I-P) ANT; (Q-X) INO. (A-D,I-L,Q-T) wildtype; (E-H,M-P,U-X) nzz-2. (B,C) Very little to no staining can beseen in the nucellus at this stage (arrows). (F,Gb,Hb) There isuniform labeling throughout the distal half of the developing ovule(arrows). (N,O) Expression is detected throughout the distal half ofthe primordium. A focus of stronger ANTexpression is seen(arrows). In N, the color reaction was stopped early in the experiment(i.e. intentionally underdeveloped). (P) ANTexpression in thedeveloping integuments is observed (arrow). (T) Note the absence ofINO expression in the inner (adaxial) cell layer of the outerintegument (arrow) and the distal corner cell of the abaxial cell layer(arrowhead). Abbreviations: ii, inner integument; fu, funiculus; mmc,megaspore mother cell; oi, outer integument. Scale bars, 20µm.

Fig. 7.A genetic model detailing aspects of NZZfunction duringovule ontogenesis. The distal, central and proximal domains areindicated (red, green and brown, respectively). D and E representdistal and epidermal factors, respectively. The brackets mark thepostulated events taking place in the absence of the repression ofBEL1and ANTby NZZin the distal region. The braces indicate thatthe relationship between the genes is not known. The question markand the dashed line indicate the unknown and probably indirecteffect of NZZon INOrepression in the developing funiculus.

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In wild type, INORNA can first be seen around stage 2-IIin about 2 epidermal cells (Fig. 6R). This pattern quicklybroadens to encompass about 4-5 epidermal cells. These cellswill be part of the abaxial (outer) cell layer of the developingouter integument around stage 2-III-IV (Fig. 6S,T).Interestingly, the tip cell, in a cross section, does not show INOlabel (Fig. 6S,T). This indicates that INO may have a functionin establishing the adaxial-abaxial polarity within the outerintegument that is separate from its proposed function insetting up adaxial-abaxial polarity within the chalaza(Villanueva et al., 1999). In nzz-2mutants the patternresembles wild type (Fig. 6U-X). However, it is likely to bedisplaced distally by a few cells since the outer integumentinitiates more distally in nzz-2(Fig. 1F,N) (see Discussion).The displacement would be quite difficult to detect in in situhybridization experiments using sectioned material.

DISCUSSION

Our genetic and molecular analysis indicates that NZZis arepressor and plays a central role in anther and several aspectsof ovule development (Fig. 7). The phenotypes of our nzzmutants deviate from the reported phenotype of the spl mutant(Yang et al., 1999). In ovules of the single spl mutant that wasisolated the alterations in the integuments and the funiculuswere not observed. In addition, some late aberrant growth ofepidermal cells of the nucellus occurs in splmutants that wedid not detect in our nzzmutants. We assume that the observeddiscrepancies are essentially due to allele-specific effects or thegenetic background. All of the three different nzz mutantsisolated in our lab show, to various degrees, the phenotypesdescribed in this paper. In addition, the observed phenotypesof the nzzmutants correlate well with the expression pattern ofNZZ (Schiefthaler et al., 1999).

NZZ antagonizes ANT, BEL1 and INO activity duringthe formation of the nucellusThe reduction of the nucellus and the failure to develop a MMCindicates that NZZhas an early function in the formation of thenucellus and/or the MMC. The mutant phenotype and thebroad expression pattern raised the possibility that NZZisrequired for the early formation of the nucellus and that theabsence of a MMC is a secondary defect (Schiefthaler et al.,1999). As an alternative explanation for the reduction of thenucellus it had been suggested that NZZ is primarily requiredfor early megasporogenesis and that nucellar ontogenesisdepends on correct MMC formation (Schiefthaler et al., 1999;Yang et al., 1999). The data presented above shed some morelight on NZZ function during nucellus development. Theyprovide supporting evidence for the former model withoutexcluding an additional role for NZZ in MMC development.

The double mutant analysis indicates that a main functionof NZZ in the formation of the nucellus consists ofantagonizing BEL1 and ANT activities in the distal region atlate stage 1 prior to the onset of megasporogenesis. EctopicBEL1and ANTactivity is detrimental to nucellus developmentwith ectopic ANTfunction leading to more severe defects (seebelow). Interestingly, the transcription of both genes ceases inthe nucellus at stage 1-II in wild type (Elliott et al., 1996;Reiser et al., 1995; Fig. 6B,C,J). Could NZZ antagonize ANT

and BEL1 by being responsible for their transcriptionalrepression in the developing nucellus? Within its limits ofresolution our analysis suggests that ovule primordia from wildtype and nzz-2mutants are of comparable PD extension at theend of stage 1 (Fig. 1A,B,E,F). In addition, the expressiondomains of BEL1and ANTare broader and extend to the distaledge of the primordium at stage 1-II/2-I. Thus, these findingsraise the possibility that NZZis a negative regulator of BEL1and ANT transcription in the incipient nucellus at stage 1-II/2-I. At present it is unclear how direct this regulation is. Giventhat NZZ is expressed in the distal region around late stage1/early stage 2 and that the putative NZZ protein showsfeatures of a transcription factor (Schiefthaler et al., 1999;Yang et al., 1999) and localizes to the nucleus (Yang et al.,1999) we would like to propose that NZZ acts as atranscriptional repressor of ANTand BEL1. Of course, furtherexperiments are needed to clarify this issue. Taking intoconsideration the early ubiquitous wild-type NZZ expression,the data also indicate that additional factors must be involvedin controlling ANTand BEL1expression distally. Furthermore,in contrast to the situation with BEL1the interactions of ANTand NZZ are more complex. Besides the ectopic distalexpression at stage 1-II, there is a focus of stronger epidermalANTexpression at an abaxial position, close to where the outerintegument develops, which is not found in wild type (Fig.6N,O). We believe this pattern is reflecting a function of NZZduring integument development (see below).

How does INOfit into the picture? The phenotype of nzz inodouble mutants suggests that NZZ is also a repressor of INOinthe distal region and that ectopic INOactivity leads to a failurein nucellus formation as well. We believe that the simultaneousmisexpression of BEL1and ANTprobably leads to the ectopicactivity of INO, since rendering ANTexpression aloneindependent of NZZactivity, by putting ANTunder the controlof the ubiquitously active 35S CaMV promoter, does not resultin a reduction of the nucellus (Krizek, 1999; Mizukami andFischer, 2000). Furthermore, we have found that at early stage2, INO RNA is absent in antand bel1mutants (S. B. and K. S.,unpublished data). Still, INO continues to be expressedexclusively in a few abaxial epidermal cells. The homeoboxgene ATML1 could be a possible candidate for providingepidermis-specific activity (Lu et al., 1996) in this process. Howdoes INO control nucellus development in nzzmutants? Wehypothesize that INOacts in a non cell-autonomous fashion andinfluences the development of the subepidermal region adjacentto the early INOexpression domain at a stage before the outerintegument is discernible as an outgrowth. This could alsoexplain the funicular phenotype of nzz inodouble mutants (seebelow). Non cell-autonomous INOfunction could be restrictedto nzzmutants but we think it likely to occur also in wild typesince it could well explain the early formation of an adaxialbulge, located within the chalaza, in inomutants (Baker et al.,1997; Schneitz et al., 1997; Villanueva et al., 1999; Fig. 3K).

In summary we outline one possible model that rationalizesour findings (Fig. 7). A key aspect is that NZZ functions as anegative regulator of ANTand BEL1in the distal region of theovule primordium. If NZZfunction is absent around stages 1-II/2-I co-misexpression of BEL1and ANTin the distal domain,in concert with additional factors, leads to the ectopicactivation of INOat a slightly more distal position within theepidermis. INO indirectly influences the development of the



tissue neighboring its expression domain, leading to a chalazalrather than a nucellar specification, thus resulting in a ‘distalshift’ of the chalaza at the expense of the nucellus. In nzz inodouble mutants ANT and BEL1would still be ectopicallyexpressed in the distal tip; however, this expression, due to theabsence of INOactivity, does not result in the chalaza ‘shiftingdistally’ and the replacement of nucellar tissue, rather, itinterferes with later aspects of nucellar differentiation.Compared to BEL1, ANTwould be the factor with a higherimpact in negatively influencing further nucellar differentiationsince plants defective for NZZand ANTfunction have a muchlarger proportion of nucelli with a MMC than nzz bel1doublemutants. Alternatively, it is possible that NZZ has a functionduring MMC differentiation or meiosis as well or thatadditional, as yet unknown factors, come into play.

NZZ and BEL1 share overlapping functions inspecifying the central regionIn nzz bel1double mutants the chalaza seems at least partiallysubstituted by a funiculus, as indicated by the epidermalmorphology at the corresponding PD position. The moleculardetails of the interaction between NZZand BEL1in the centraldomain are presently unclear. However, the defect can bedescribed as a homeotic phenotype with the chalaza replacedby a funiculus (or a pattern duplication of the proximaldomain) indicating that NZZand BEL1are required for thespecification of the chalaza and thus represent chalaza identitygenes. Since the nzzand bel1alleles used are recessive loss-of-function mutations it appears that in wild type, NZZ andBEL1 somehow direct a developmental pathway towardschalazal identity, which otherwise, perhaps by default, wouldlead to funicular identity.

The role of NZZ in integument developmentThe ovules ofnzzmutants often exhibit a reduced growth ofboth integuments indicating that NZZhas a function inintegument development (Fig. 1G,H). This is supported by thefact that elevated levels of NZZexpression can be found in bothinteguments starting around the time of their inception(Schiefthaler et al., 1999; Fig. 5B-D). The genetic analysissuggests that antis essentially epistatic to nzzas far asintegument development is concerned (Fig. 7). With respect toouter integument ontogenesis this holds true for ino as well.Interestingly, in antmutants the elevated NZZexpression in theentire chalazal epidermis is not observed, while in ino mutantsspecifically the elevated NZZexpression in the outerintegument domain of the chalaza is not detected. Thus, itappears that during the initial phase of integument developmentNZZ functions downstream of ANTand INO. Furthermore,ANT and INO act as transcriptional regulators of NZZin thisprocess with ANTfunctioning during the development of bothinteguments and INObeing specific for outer integumentontogenesis. Both genes are not required for basal level of NZZtranscription, however.

One of the functions of NZZ in integument developmentcould again be in repressing ANTactivity. This hypothesiscould explain the focus of stronger epidermal ANTexpressionin young ovule primordia of nzz mutants. In addition,constitutive overexpression of ANTin the Ler backgroundleads to a reduction of the integuments comparable to thereduction observed in nzzmutants (Krizek, 1999). If true it

lends support to the idea that proper integument developmentdepends on the precise level of ANTactivity (Krizek, 1999).

How do ANTand INOrelate to each other during early outerintegument development? Genetic experiments indicated thatant is epistatic to ino(Baker et al., 1997) but it is presentlyunclear how this is reflected at the molecular level. In any case,both ANTand INO encode putative transcription factors (Elliottet al., 1996; Klucher et al., 1996; Villanueva et al., 1999) andit will be interesting to explore whether the two genes controlNZZ transcription directly.

The role of NZZ during funiculus developmentThe funiculus of a nzz mutant ovules shows hyperplasticgrowth since the funiculus is elongated due to a larger cellnumber but the overall morphology seems not affected (Table1; Fig. 1G,H). The hyperplastic growth in the funiculus issuppressed in nzz antand nzz inodouble mutants indicatingthat NZZ functions as a repressor of those two genes duringfuniculus development. This interpretation fits well withrespect to ANT,since overexpressing ANTin the Lerbackground essentially leads to a phenocopy of the nzzphenotype in the funiculus (and the integuments, see above.Compare Fig. 1G,H with figure 3N-P in Krizek, 1999).Unfortunately, due to the absence of suitable markers, itremains an open question whether or not the funicularrepression of ANTby NZZoccurs at the transcriptional level aswell. It often appears as if the expression domain of ANTinthe distal part of the funiculus at early stage 2 is somewhatbroader in nzzmutants than in wild type. However, we foundit difficult to compare the exact position of the proximalboundary (or the level) of the ANTexpression in funiculi ofwild type and nzzmutants at later stages. This was in part dueto the fact that it was difficult to establish whether or not thevery proximal base of the funiculus was stained.

The genetics suggest that NZZ is a repressor of INOduringfuniculus formation. How this is achieved remains at presentdifficult to assess. One possible explanation of the nzz inofunicular phenotype is that INO renders the neighboringdeveloping funicular cells competent to respond to elevatedANT levels caused by the absence of wild-type NZZ activity.Experiments are underway that address this issue in moredetail.

NZZ couples PD patterning and cell proliferationduring ovule primordium formationSince there is generally no cell movement in plants,organogenesis essentially results from the patterned control ofcell division and cell shape changes (Meyerowitz, 1997). Howis this achieved during ovule development? NZZ and BEL1have properties of chalaza identity genes. In addition, NZZnegatively regulates BEL1,in the nucellus, and the cell numbercontrol gene, ANT,in the nucellus, the funiculus and probablythe developing integuments. Thus, there are aspects of NZZfunction related to conferring identity and to controlling cellnumber. It suggests that NZZ links PD patterning and thecontrol of cell proliferation during ovule development. Furtheranalysis of NZZshould reveal additional details about theunderlying molecular mechanism.

We thank U. Jauch for help with the SEM and J.-J. Pittet for theartwork. We also thank David Chevalier, Ursula Schiefthaler and

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Patrick Sieber for stimulating discussions and Martin Hülskamp forcomments on the manuscript. This work was supported by the SwissNational Science Foundation (grant 31-53032.97) and by the Kantonof Zürich.

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