Sex Plant Reprod (2010) 23:115–121 DOI 10.1007/s00497-009-0130-3
ORIGINAL ARTICLE
Genetic interaction between AINTEGUMENTA (ANT) and the ovule identity genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) Alessia Losa • Monica Colombo • Vittoria Brambilla Lucia Colombo
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Received: 11 June 2009 / Accepted: 15 December 2009 / Published online: 30 December 2009 Ó Springer-Verlag 2009
Abstract AINTEGUMENTA (ANT) promotes initiation and growth of ovule integuments which cell fate is specified by ovule identity factors, such as SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2). To study the genetic interaction between ANT and the ovule identity genes, we have obtained a stk shp1 shp2 ant quadruple mutant. The molecular and morphological characterization of the quadruple mutant and its comparison with the stk shp1 shp2 triple mutant, the shp1 shp2 ant triple mutant and the stk ant double mutant are here presented. Keywords ANT Ovule development Ovule identity genes SHP1 SHP2 STK
Introduction The ovule, a specialized structure that develops inside the carpel, is the site where processes essential for sexual plant
Communicated by Ueli Grossniklaus. A. Losa M. Colombo V. Brambilla L. Colombo (&) Dipartimento di Biologia, Universita` degli Studi di Milano, 20133 Milan, Italy e-mail:
[email protected] Present Address: A. Losa Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita` degli Studi di Milano, 20133 Milan, Italy Present Address: V. Brambilla Max Planck Institute for Plant Breeding, Carl von Linne Weg 10, 50829 Cologne, Germany
reproduction, such as megasporogenesis, megagametogenesis, fertilization and embryogenesis, occur. Along the proximal–distal axis of the ovule primordia, three regions can be identified. The proximal region differentiates into the funiculus, which connects the ovule to the placenta. The central chalazal region forms two integuments that grow asymmetrically to enclose the nucellus, leaving a small opening, the micropyle, which allows the pollen tube to enter the embryo sac. The distal portion of the ovule primordium will form the nucellus, within which the female gametophyte will develop. Female gametophyte formation occurs over two phases referred to as megasporogenesis and megagametogenesis (Schneitz et al. 1995; Christensen et al. 1997). During megasporogenesis, the diploid megaspore mother cell goes through meiotic division, forming a tetrad of haploid megaspores of which only the chalazal one survives. During megagametogenesis, the embryo sac develops from the functional megaspore by undergoing three mitotic divisions and subsequent cellularization. The molecular control of ovule development has been widely studied in Arabidopsis and other plants, such as rice and petunia. In the last several years, the characterization of a large number of ovule defective mutants has contributed to a better understanding of this process, and several genes controlling ovule development have been identified (for review see Colombo et al. 2008). Previous studies have demonstrated that the MADS-box transcription factor genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) redundantly control ovule integuments identity (Pinyopich et al. 2003; Brambilla et al. 2007). In the stk shp1 shp2 triple mutant, the integuments are converted into carpel-like structures, whereas in single or double mutant combinations of these genes, ovule identity is not affected. Furthermore, ectopic expression of one of these genes results
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in ovule formation of sepals (Favaro et al. 2003; Pinyopich et al. 2003; Battaglia et al. 2006). In the stk single mutant, as well as in the stk shp1 shp2 triple mutant, the funiculus is longer and thicker, due to the role of STK in controlling cell expansion and cell division in this structure (Pinyopich et al. 2003). Another important gene that plays fundamental and multiple roles in ovule formation and development is AINTEGUMENTA (ANT), which encodes an AP2-like factor. In the ant mutant, the number of ovule primordia is reduced in respect to the wild type. Furthermore, ovule integuments fail to develop from the chalaza, and megasporogenesis does not proceed after meiosis (Elliott et al. 1996; Klucher et al. 1996; Baker et al. 1997). The expression domains of all these genes are partially overlapping during ovule development. The SHP genes have identical expression patterns. SHP genes are initially expressed broadly in the gynoecium primordium, but soon their expression becomes restricted to the valve margins and also in the developing ovules (Savidge et al. 1995; Flanagan et al. 1996). STK is uniformly expressed in placental tissues and ovule primordia. In mature ovules, STK is expressed strongly in the funiculus and weakly in integuments (Rounsley et al. 1995; Pinyopich et al. 2003). ANT is highly expressed in the placenta and in the arising ovule primordia. In the developing ovule, its expression becomes localized to the chalaza and subsequently to the integuments that arise from this region. Here, we describe the genetic interaction between the ovule identity genes and ANT. The stk shp1 shp2 ant quadruple mutant ovule lacks integument development as in ant single mutant. However, the length of such ovules is increased significantly respect to ant single mutant, suggesting that these genes might redundantly control cell expansion during funiculus development.
Materials and methods Plant material Plants were grown at 21°C under short-day (8 h light/16 h dark) or long-day (16 h light/8 h dark) conditions. The stk shp1 shp2 mutant (Columbia accession) was kindly provided by Yanofsky; ant-4 mutant seeds (Landsberg erecta accession, Baker et al. 1997) were obtained from the Nottingham Arabidopsis Stock Centre. The stk shp1 shp2 ant quadruple mutant was obtained by pollinating the STK/ stk shp1 shp2 triple mutant with ant-4 homozygous pollen. The quadruple mutant was maintained by selfing the STK/ stk shp1 shp2 ANT-4/ant-4 plants obtained in the segregating population. All the mutant combinations were analyzed in F2, F3 and F4 populations. Twenty stk shp1 shp2
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ant quadruple mutant plants were analyzed (5 pistils per plant). Analysis of STK expression in the stk shp1 shp2 ant quadruple mutant To monitor STK expression, the STK::GUS reporter line (Kooiker et al. 2005) was crossed to the stk shp1 shp2 ant mutant and the resulting F2 segregating population was analyzed. Tissue for GUS staining was prefixed in ice-cold 90% acetone at -20°C for 1 h, rinsed with 50 mM phosphate buffer (pH 7.0) three times for 5 min, placed in staining solution (1 mg ml-1 X-Gluc, 0.1% Triton X-100, 2 mM Fe2?CN, 2 mM Fe3?CN, 50 mM phosphate buffer pH 7.0, 10 mM EDTA), vacuum infiltrated for 5 min and incubated at 37°C for 24 h. After staining, the tissue was cleared in 70% ethanol, then mounted in chloral hydrate/ glycerol/water solution (8:1:2) and observed using a Zeiss Axiophot D1 microscope equipped with Nomarski differential interference contrast (DIC) optics. Mutant genotyping Genotyping of stk-2, shp1-1, shp2-1 and ant-4 alleles has been performed as described elsewhere (Brambilla et al. 2007; Colombo et al. 2009). Morphological analysis by differential interference contrast microscopy (DIC) and aniline blue staining To analyze ovule development in the mutant plants, flowers at different developmental stages were cleared in a chloral hydrate/glycerol/water solution (8:1:2). Pistils were dissected under a stereomicroscope and subsequently observed using a Zeiss Axiophot D1 microscope with DIC imaging. Images were recorded with an Axiocam MRc5 camera (Zeiss) using the Axiovision program (version 4.1). To observe callose deposition in megaspore mother cell and tetrads, ovules were cleared, immersed in an aniline blue solution (Motamayor et al. 2000) and observed by fluorescence microscopy using 40 , 6-diamino-phenylindole (DAPI) excitation and emission filters. Morphological analysis by scanning electron microscopy Plant material was fixed at 4°C overnight in 50% ethanol, 5% acetic acid and 3.7% formaldehyde in 0.025 M phosphate buffer, pH 7.0. Samples were subsequently washed in two 20-min changes of 70% ethanol in 0.025 M phosphate buffer pH 7.0. Ovules were extracted from pistils and incubated in the same solution. After dissection, isolated ovules were rehydrated through an ethanol series and then
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postfixed for 4 h in 1% osmium tetroxide in 0.05 M phosphate buffer pH 7, dehydrated gradually in an ethanol series to 100% ethanol and dried in liquid carbon dioxide using critical point drying. Samples were subsequently coated with gold using a sputter coater (SEMPREP2; Nanotech) and observed with a LEO 1430 scanning electron microscope (LEO Electron Microscopy). In situ hybridization analysis Arabidopsis flowers were fixed and embedded in paraffin as described previously (Brambilla et al. 2007). Sections of plant tissue were probed with digoxigenin-labeled STK and SHP1 antisense RNA. The antisense STK probe corresponds to nucleotides 455–818. The cDNA sequence without the MADS-box and K-box domain was used for SHP1. The probe was subsequently hydrolyzed in carbonate buffer at pH 10.2 for 60 min. Hybridization and immunological detection were both performed as described by Brambilla et al. (2007).
Results The stk shp1 shp2 ant quadruple mutant ovule lacks integuments and has a longer funiculus In wild-type plants (Fig. 1a–e), ovule primordia arise from the placenta as finger-like protrusions with radial symmetry at stage 9 of flower development (Smyth et al. 1990). At stage 10, the primordia elongate and the patterning process occurs along the proximal–distal axis so that funiculus, chalaza and nucellus can be recognized. At stage 11, the inner and outer integuments start to develop from the chalaza (Fig. 1a, b) and megasporogenesis takes place in the nucellus (Fig. 1c). Megagametogenesis and integument development proceed concomitantly during stage 12. Both integuments continue to grow around the nucellus. By the end of stage 13, the outer integument has completely overgrown the inner integument and nucellus, leaving only a small opening for pollen tube penetration at the micropyle (Fig. 1e; Schneitz et al. 1995). In the ant-4 mutant (Fig. 1k–n), as already described by Klucher et al. (1996) and Baker et al. (1997), the number of ovule primordia is drastically reduced with respect to wild type (Fig. 1n), and the integument primordia do not initiate development, whereas the funiculus seems apparently normal (Fig. 1k). Detailed analysis of the stk shp1 shp2 mutant (Fig. 1f–j) shows that the ovule ontogenesis process proceeds similar to that of wild-type plants until stage 12, as has been described by previous studies (Brambilla et al. 2007; Battaglia et al. 2008). In the triple mutant, ovule primordia morphology (compare Fig. 1f, b) as well as their number
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(compare Fig. 1j, e) is similar to wild type. Moreover, megasporogenesis is not affected (Fig. 1g; Battaglia et al. 2008). Differences compared to wild type are visible at later developmental stages, starting from mid-stage 12. Several ovules in the triple mutant have integuments that have converted into carpel-like structure (Fig. 1h), as previously described (Pinyopich et al. 2003; Brambilla et al. 2007; Battaglia et al. 2008). However, other types of integument defects can also be observed in the triple mutant. The growth of the outer integument is often reduced (10% of the total ovules observed) and in a very few ovules (2% of the total ovules observed), the integuments do not develop at all (Fig. 1i), giving rise to a structure that resembles the ant mutant ovule. These antlike ovules show a very long funiculus, probably due to the mutation in stk. The observation that ant-like ovules occasionally develop in the stk shp1 shp2 triple mutant and the observation that ANT expression partially overlaps with the ovule identity genes suggests a possible functional interaction between SHP1, SHP2, STK and ANT. To investigate this, a stk shp1 shp2 ant quadruple mutant was generated by crossing the STK/stk shp1 shp2 triple mutant with the ant-4 mutant. A large number of mutant combinations were obtained in the F2 population, in which all alleles segregated. To analyze the ovule defects and the developmental events in these mutants, optical and scanning electron microscopy analyses were performed on different specific mutant combinations from plants belonging to F2, F3 and F4 populations. Whereas in the shp1 shp2 ant triple mutant (Fig. 1o–p), the ovules develop as in the ant single mutant, the ovule phenotype of the stk ant double mutant is clearly additive (Fig. 1q–u). This mutant develops ovules displaying an enlarged funiculus specific to the stk single mutant, and they lack integument development as observed in the ant-4 single mutant (Fig. 1s). Furthermore, in the stk shp1 shp2 ant quadruple mutant (Fig. 1v–z), the overall length of the ovule increases significantly (Fig. 1v, x), probably because cells forming the funiculus (Fig. 1y) are much larger than those of wild type (Fig. 1d) and all the other mutant combinations (Fig. 1i, k, o and s). Besides the defects in ovule development, morphological analysis revealed that the shp1 shp2 ant (Fig. 1p) and the stk shp1 shp2 ant mutant gynoecia frequently fail to fuse at the apical region (Colombo et al. 2009). Callose deposition in ovules is a marker associated with cells undergoing megasporogenesis and can be visualized by staining with blue aniline (Schneitz et al. 1995). In wild-type ovules, callose accumulation appears during meiosis shortly before cytokinesis and becomes concentrated at the site of the newly formed cell plate. Subsequently, callose accumulates
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toward the micropylar end and in the degenerating spores. As megagametogenesis begins, callose disappears from the functional megaspore (Tucker et al. 2001). However, megagametogenesis did not occur in stk shp1 shp2 mutants (Fig. 1g), as well as ant-4 single mutants (Fig. 1m), the stk ant (Fig. 1r) and the stk shp1shp2 ant mutants (Fig. 1w), although megasporogenesis proceeded as in wild-type ovules (Fig. 1c) until the formation of the tetrad.
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b Fig. 1 Ovule development in wild type (a–e), stk shp1 shp2 triple mutant (f–j), ant single mutant (k–n), shp1 shp2 ant triple mutant (o–p), stk ant double mutant (q–u) and stk shp1 shp2 ant quadruple mutant (v–z). Scanning electron microscopy was used in a, d–e, h–k, n–p, s–u, and x–z. b, f, l, q and v are cleared whole mounted ovules observed using Nomarski differential interference contrast (DIC) microscopy. c, g, m, r and w are the corresponding whole mounted ovules treated to observe callose deposition in the newly formed cell walls via aniline blue fluorescence. a Wild-type ovule primordia showing the developing inner and outer integuments. b The outer integument displays asymmetric growth and, by the end of stage 13, it will cover completely the nucellus and the inner integument. c The developing ovule in b. Megasporogenesis takes place in the nucellus: blue aniline staining reveals the formation of the four haploid megaspores. d Close-up of wild-type funiculus in mature ovules. The micropyle (mp) can be seen in the lower ovule. e Mature wild-type ovules inside the carpel. f In the stk shp1 shp2 triple mutant, the ovule primordium develops normally until stage 11. The primordium arises from the placenta and the patterning along the proximal–distal axis is correctly established. The integument primordia differentiate. g The blue aniline staining marks the meiotic division in the nucellus. Megasporogenesis therefore is not affected in the triple mutant. h Integument development is affected starting from stage 12. In most of the ovules, integuments do not cover the nucellus and form carpel-like structures. i ant-like ovules, lacking integuments and with a very long funiculus, can occasionally develop from the placenta. j stk shp1 shp2 mature pistil showing the ovules inside. k In the ant-4 mutant, ovule primordia develop as in the wild type until stage 11. During this stage, integuments do not initiate development, as already described by Klucher et al. (1996). At maturity, the ovules lack the integuments and the nucellus develops as a thin, elongated structure, whereas the funiculus is apparently normal. l Cleared ant mutant ovules observed using Nomarski differential interference contrast (DIC) microscopy. m Aniline blue staining of the ovules in l. In the upper ovule, megasporogenesis is taking place. n At later stages of growth, bulbar epidermal cells develop in the chalazal region where integuments normally arise. As a result, the mature ovule shows a bending in the region corresponding to the integument–nucellus boundary, as already observed in ant-9 (Elliott et al. 1996). Ovule number is reduced compared to the wild type. o Close-up of a shp1 shp2 ant mutant ovule. In this triple mutant, ovules develop as in the ant single mutant. p Mature triple mutant pistil. Ovule number reduction is visible as well as the fusion failure in the apical region of the gynoecium. q stk ant double mutant ovule primordia. Nucellus and funiculus differentiate at stage 11. Integuments primordia do not develop from the chalaza. r Blue aniline staining of the ovule primordia in q showing megasporogenesis in the nucellus. s Close-up of a stk ant double mutant ovule. t Close-up of mature double mutant ovules. Integuments are lacking. The three different regions (funiculus, chalaza, nucellus) are not clearly distinguishable. u stk ant ovules in a mature pistil. Ovule number is comparable to ant single mutant. v stk shp1 shp2 ant quadruple mutant ovules. Ovule length is clearly increased. w Meiosis normally occurs in the nucellus of the quadruple mutant implying that megasporogenesis is not affected in the stk shp1 shp2 ant mutant. x A quadruple mutant ovule showing no integument formation. The three regions forming the ovule (funiculus, chalaza and nucellus) are not clearly distinguishable. y Close-up of the bigger cells forming the funiculus of the quadruple mutant ovule in x. z stk shp1 shp2 ant ovules in a mature pistil. n nucellus, ii inner integument, oi outer integument, f funiculus, c chalazal region, mp micropyle. Scale bars o 20 lm; a, d, k, s, t, y 25 lm; b, c, e, f, g, h, i, l, m, q, r, v, w 50 lm; u, x, z 60 lm; n 100 lm; j, p 150 lm
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b Fig. 2 Spatial expression of ANT, STK and SHP1 in wild type and different mutant combinations. In situ hybridization, analysis for ANT expression in the stk shp1 shp2 triple mutant using a digoxigeninlabeled antisense RNA probe (a–c). In situ hybridization to analyze SHP1 expression in wild type (d–e) and ant-4 single mutant (f–g). In situ hybridization to analyze STK expression in wild type (h–i) and ant-4 single mutant (j–k). Dark-field microscopy was used in a–c. Nomarski differential interference contrast (DIC) microscopy was used in d–k. a ANT expression in a stk shp1 shp2 triple mutant flower. b The hybridization signal is visible in placenta and ovule primordia. c ANT is expressed strongly in the carpelloid structures typical for the triple mutant. d SHP1 expression profile in a wild type developing carpel and in e mature ovules. f SHP1 mRNA detected in ovule primordia in the ant-4 single mutant. g Later in development, SHP1 expression becomes limited, in the ant-4 ovule, to the chalazal region. h STK expression in the placenta and in the emerging ovule primordia. i In the mature ovule STK expression is restricted to the funiculus and the integuments. j STK mRNA is strongly expressed in ant-4 ovule primordia. k In mature carpel, STK is expressed in the funiculus and in the chalaza region where the integuments normally arise. While no signal is detected in the nucellus, some hybridization appears in the gynoecium wall. p placenta, o ovule primordium, cl carpel-like, en endothelium, n nucellus, ch chalaza, f funiculus. Scale bar 50 lm
Gene expression analysis in the mutant combinations The expression domains of ANT and the ovule identity genes STK, SHP1 and SHP2 partially overlap during ovule development. In order to understand whether these genes are able to regulate each others’ expression, in situ hybridisation and reporter line studies were performed. First, we analyzed ANT expression in the stk shp1 shp2 triple mutant. In the wild-type carpel, ANT is strongly expressed in the placenta and in the arising ovule primordium. Later, its expression becomes restricted to the chalaza and the developing integuments. At stage 13 of flower development, expression is found in the chalaza and in the
endothelium, which is derived from the inner integument. ANT expression occurs in the distal half of the funiculus throughout ovule development (Elliott et al. 1996). In situ hybridization showed that ANT expression is not changed in the stk shp1 shp2 triple mutant (Fig. 2a, b). Furthermore, ANT expression was also observed in the carpel-like structures that develop instead of ovule integuments, which are characteristic for the triple mutant (Fig. 2c). To investigate whether ANT is involved in the regulation of SHP1, SHP2 or STK expression, in situ hybridization experiments were performed using the ant single mutant. SHP1 (Flanagan et al. 1996) and SHP2 (Savidge et al. 1995) share an almost identical expression pattern. They are initially detected throughout the developing gynoecium. At stage 10, their expression extends to the valve margins, replum, septum and developing ovules. Soon thereafter, the expression becomes limited to the valve margins and the ovules (Fig. 2d, e). The same expression pattern is retained in the ant-4 single mutant during all stages of gynoecium development (Fig. 2f, g). STK expression is first detected in placental tissue and young ovule primordia (Fig. 2h). In the mature ovule, at stage 13, its expression becomes restricted to the funiculus and the integuments (Fig. 2i), as described by Pinyopich et al. (2003). Analysis of STK expression in the ant-4 mutant showed that STK mRNA was detected in the placenta and in the ovule primordia (Fig. 2j), as well as in the mature mutant ovule (Fig. 2k), similar to the expression profile in wild-type plants. To study STK expression in the stk ant double mutant and in the stk shp1 shp2 ant quadruple mutant, we crossed a STK::GUS reporter line (previously described by Kooiker
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Fig. 3 Expression of the STK::GUS reporter line in the wild type (a) and in the segregating offspring population of the STK/stk SHP1/shp1 SHP2/shp2 ANT/ant mutant (b–e). The ovules shown here are from middle to late stage 2 according to Schneitz et al. (1995). a GUS activity is strongly detected in the placenta and in the developing ovule, in the funiculus and in the inner and outer integument. b In the stk ant double mutant, as well as in c the ant single mutant, staining can be seen in the placenta and in the developing mutant ovule, in the regions corresponding to the funiculus and the chalaza. d In the stk single mutant, as well as in e, the stk shp1 shp2 triple mutant, the GUS staining pattern is similar to the wild type. Scale bar 50 lm
et al. 2005; Brambilla et al. 2007), with the quadruple mutant, and we analyzed the F2 segregating population (Fig. 3). Histochemical analysis of GUS expression showed that in the single mutants ant (Fig. 3c) and stk (Fig. 3d), as well as in the double mutant stk ant (Fig. 3b) and in the triple mutant stk shp1 shp2 (Fig. 3e), the STK promoter is activated as in wild-type plants (Fig. 3a). The same GUS staining pattern can be also observed in the stk shp1 shp2 ant quadruple mutant (data not shown).
Discussion STK, SHP1 and SHP2 are MADS-box transcription factor genes that belong to the AGAMOUS subfamily and play specific roles during carpel and ovule development (Pinyopich et al. 2003). STK regulates cell expansion and cell division in the funiculus, and it is therefore required for normal development of this structure and for dispersal of the mature seeds (Pinyopich et al. 2003). SHP1 and SHP2 redundantly specify valve margin formation (Liljegren et al. 2000) and can substitute for the AG function in carpel specification (Pinyopich et al. 2003). The double mutant
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shp1 shp2 does not exhibit altered ovule development; however, when these genes are combined together in a stk shp1 shp2 triple mutant, normal ovule development is severely disrupted, with some of the ovules converted into carpelloid structures (Pinyopich et al. 2003), implying that these genes share a redundant function in ovule development. In particular, it has recently been demonstrated that STK, SHP1 and SHP2 interact with the homeobox protein BEL1 to regulate integument identity determination and development (Brambilla et al. 2007). A crucial factor in controlling the initiation of integument development is AINTEGUMENTA (ANT), a transcription factor belonging to the AP2 family (Elliott et al. 1996; Klucher et al. 1996; Baker et al. 1997; Gross-Hardt et al. 2002). This gene regulates organ initiation and organ size by maintaining the proliferative potential of organ primordia (Elliott et al. 1996; Klucher et al. 1996; Krizek 1999; Mizukami and Fischer 2000). In addition, it is involved in the regulation of floral organ identity and organ polarity in combination with YABBY genes (Nole-Wilson and Krizek 2006). ANT plays also multiple roles during ovule initiation and development. In particular, mutations in ANT result in a failure of the ovule to initiate integument formation (Baker et al. 1997; Elliott et al. 1996; Klucher et al. 1996). The observation that ant-like ovules can occasionally develop in the stk shp1 shp2 triple mutant, together with the quite similar expression pattern in the ovule, suggests a possible functional interaction between SHP1, SHP2, STK and ANT. To study this interaction during ovule development, we combined the stk shp1 shp2 triple mutant with the ant-4 mutant. The observed ovule phenotypes were consistent in the F2, F3 and F4 segregating populations and in the population derived from the cross with the STK::GUS reporter line, indicating that phenotypic effects are not the result of differences in the accession background of our mutants. We observed that the stk ant double mutant is additive, since double mutant ovules have an enlarged funiculus, which is typical of the stk single mutant, and they lack integument development as in the ant single mutant. A similar phenotype can be observed in the stk shp1 shp2 ant quadruple mutant ovules. These data suggest that ANT acts in an independent pathway with respect to the ovule identity genes. This finding is strengthened by the fact that ANT expression does not require ovule identity gene activity, as demonstrated by in situ hybridization experiment using the stk shp1 shp2 triple mutant. Likewise, in situ hybridization experiments showed that the ovule identity genes SHP1 and STK are normally expressed in the ant single mutant. In order to better study STK expression regulation, the stk shp1 shp2 ant quadruple mutant was crossed with a STK::GUS reporter line, and the segregating offspring population was analyzed.
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Wild-type STK promoter activity was identified in all the different mutant combinations, confirming that STK expression is not dependent on ANT activity and suggesting that probably the ovule identity genes, or at least STK, are not part of an autoregulatory loop. However, in the stk shp1 shp2 ant quadruple mutant, the cells forming the funiculus are much larger than the wild type, and the total length of the ovule is significantly increased, suggesting that ANT, which is expressed in the distal half of the funiculus throughout ovule development (Elliott et al. 1996), and the ovule identity genes might play a redundant function in controlling funiculus development. Acknowledgments We thank Martin Yanofsky for providing the stk shp1 shp2 triple mutant. Monica Colombo is supported by the University of Milan. Scanning electron microscopy analysis was performed with the help of Giulio Melone at the Centro Interdipartimentale Microscopia Avanzata, an advanced microscopy laboratory established by University of Milan. We thank Martin Kater for critical reading of the manuscript. Funding has been provided by ERA-PG (MIUR RBER062B5L).
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