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The Plant Journal (2004) 37, 707±719 doi: 10.1111/j.1365-313X.2003.01996.x FIE and CURLY LEAF polycomb proteins interact in the regulation of homeobox gene expression during sporophyte development Aviva Katz, Moran Oliva, Assaf Mosquna, O®r Hakim and Nir Ohad Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel Received 22 September 2003; revised 20 November 2003; accepted 26 November 2003. For correspondence (fax 972 3 640 9380; e-mail [email protected]). Summary The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40 homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and represses ¯owering during embryo and seedling development. As ®e alleles are not transmitted maternally, homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological aberrations, including loss of apical dominance, curled leaves, early ¯owering and homeotic conversion of leaves, ¯ower organs and ovules into carpel-like structures. These morphological aberrations are similar to those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf ) mutants. Furthermore, the aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I KNOTTED-like homeobox (KNOX ) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2 ) and SHOOTMERISTEMLESS (STM ), whereas BREVIPEDICELLUS (BP ) was upregulated in FIE-silenced plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological characteristics, together with the molecular and biochemical data presented in this work, strongly suggest that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our ®ndings demonstrate that the versatility of the plant FIE function, which is derived from association with different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene expression throughout the plant life cycle. Keywords: CLF, FIE, homeobox genes, KNOX genes, polycomb proteins, sporophyte development. Introduction The alternation between the sporophyte and gametophyte phases of the life cycle of higher plants requires the activation and repression of appropriate developmental programmes. FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) protein is an important regulator of reproductive programmes in plants. Mutations in the Arabidopsis FIE gene affect the female gametophyte, allowing the central cell to replicate and differentiate into endosperm, in the absence of fertilization, without triggering the development of the egg cell into an embryo (Ohad et al., 1996). FIE encodes a WD40-type protein, homologous to the Drosophila polycomb group (PcG) protein, extra sex comb (ESC) (Ohad et al., 1999). In plants, as in insects and mammals, PcGs are involved in the regulation of various developmenß 2004 Blackwell Publishing Ltd tal programmes (reviewed by Berger and Gaudin, 2003; Chaudhury et al., 1998; Goodrich and Tweedie, 2002; Hsieh et al., 2003; Kohler and Grossniklaus, 2002; Sung et al., 2003; Wagner, 2003). In insects and mammals, the WD40type protein ESC and its orthologue embryonic ectoderm development (EED) form complexes containing SET domain protein (Jones et al., 1998; Sewalt et al., 1998; Tie et al., 1998). These complexes bind to and alter chromatin condensation, resulting in downregulation of homeotic target gene expression (reviewed by Simon and Tamkun, 2002). Similarly, interactions between the Arabidopsis SET domain PcG protein MEDEA (MEA) and FIE have been shown to take place in vitro (Luo et al., 1999; Spillane et al., 2000; Yadegari et al., 2000), indicating that 707 708 Aviva Katz et al. FIE and MEA form a PcG complex in vivo, regulating endosperm and embryo development as was recently shown (Kohler et al., 2003a). FIE was found to repress the expression of MADS-box gene family members (MINICHROMOSOME MAINTENANCE 1 (MCM1) genes in yeast, AGAMOUS (AG ) in Arabidopsis, DEFICIENS (DEF ) in Antirrhinum and serum response factor (SRF ) in humans) (Riechmann and Meyerowitz, 1977), and to prevent seedlings from ¯owering precociously (Kinoshita et al., 2001). Similarly, EMBRYONIC FLOWER2 (EMF2) and VERNALIZATION2 (VRN2), homologues of Su(z)12, a subunit of the Drosophila ESC protein complex, control ¯owering through the regulation of MADS-box genes (Gendall et al., 2001; Moon et al., 2003). FIE, MEA and FERTILIZATION-INDEPENDENT SEED2 (FIS2) were shown to control expression of PHERES1 (PHE1), a type I MADS-box gene, which regulates seed development. Furthermore, FIE and MEA associate with the promoter region of PHE1 (Kohler et al., 2003b). Thus, whereas PcG proteins in plants control MADS-box genes, in mammals and insects they control homeotic genes, which belong to the homeobox gene family. The expression of FIE mRNA in all wild-type Arabidopsis tissue during the vegetative and reproductive phases (Ohad et al., 1999) implies a possible role for FIE throughout the entire plant life cycle. The question arises as to what programmes are controlled by the FIE±PcG complex in the sporophyte, and what proteins take part in such complex. Until recently, it was not possible to study FIE function in the sporophyte, as mutant ®e alleles are not transmitted through the female gamete, thus preventing the formation of homozygous mutant plants (Ohad et al., 1996). Using a pFIE:FIE-GFP (GREEN FLUORESCENT PROTEIN) transgene, it was possible to partly rescue ®e mutant embryos, which were able to germinate, but failed to develop into an adult plant. Occasionally, however, seedling shoots, hypocotyls and roots produced ¯ower-like structures and organs, demonstrating that FIE is required to suppress the ¯oral programme in the early stages of plant development (Kinoshita et al., 2001). To explore the role of FIE during sporophyte development and understand how it functions, we characterized Arabidopsis transgenic plants co-suppressed for FIE expression. Signi®cantly reduced levels of FIE protein caused pleiotropic aberrant phenotypes, while still allowing the plants to reach maturity. Analysis of the plants revealed that FIE controls leaf development by regulating members of the homeobox and the MADS-box gene families. We also found that FIE±PcG protein interacts with CURLY LEAF (CLF), a SET domain PcG protein known to regulate leaf and ¯ower differentiation (Goodrich et al., 1997). The above results imply that FIE may associate with different SET-domain proteins to form PcG complexes controlling gametophytic and sporophytic developmental programmes. Results Ectopic expression of GFP:FIE resulted in FIE co-suppression Transgenic plants were generated to express a translational fusion between GFP and FIE cDNA, driven by the cauli¯ower mosaic virus (CaMV) 35S promoter. Seven out of 60 independent transgenic T1 plants displayed similar abnormal phenotypes, including loss of apical dominance, abnormal rosette leaves, curled cauline leaves, early ¯owering, homeotic conversions of ¯ower organs and delayed senescence. Southern blot analysis of genomic DNA, extracted from T3 plants of the above seven lines, using FIE cDNA as a probe, revealed insertions into several different loci (data not shown), indicating that the observed abnormal phenotypes did not result from the disruption of a speci®c locus. T3 homozygous transgenic plants segregated to plants displaying either abnormal or normal phenotypes. Expression of the GFP-FIE transgene and endogenous FIE in the above T3 segregating plants was tested using semiquantitative RT-PCR. The expression levels of the GFP-FIE transgene were signi®cantly lower in the abnormal transgenic plants as compared to transgenic plants exhibiting the normal phenotype (Figure 1a). The expression level of the endogenous FIE was similar in wild-type plants and transgenic segregates with normal phenotypes. However, transcript level of the endogenous FIE was signi®cantly lower in the abnormal transgenic segregates (Figure 1a). The reduction in endogenous and transgenic FIE expression levels was observed in both vegetative (rosette leaves) and reproductive tissue (cauline leaves and in¯orescences). The correlation between abnormal phenotypes and reduced expression of both endogenous and transgenic FIE indicated that the abnormal phenotype was caused by co-suppression of FIE expression (Denli and Hannon, 2003; Szweykowska-Kulinska et al., 2003). Furthermore, among some of the transgenic lines, few plants displayed chimaeric phenotypes producing normal and abnormal branches. This further supports the conclusion that silencing resulted from transgene-induced co-suppression. Western analyses performed with af®nitypuri®ed antibodies against FIE have shown that FIE is not detected in the rosette leaves of abnormal transgenic plants, but does appear in wild-type leaves, con®rming the above conclusion (Figure 1b). Thus, we refer to the abnormal GFP:FIE transgenic plants as `FIE-silenced plants'. FIE-silenced plants display pleiotropic phenotypes The FIE-silenced plants exhibited a range of abnormal phenotypes as shown in Figure 2. Loss of apical dominance was evident from the formation of six to eight stems as compared to one to three stems in wild-type plants ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 FIE regulates homeobox gene expression in sporophyte development Figure 1. Expression of FIE in wild-type and FIE-silenced plants. (a) Ethidium bromide-stained gel of RT-PCR products. Total RNA extracted from wild-type (wt) and GFP:FIE transgenic plants displaying either normal (normal) or abnormal phenotypes (abnormal) served as template. RT-PCR was performed on RNA samples extracted from rosette, cauline leaves and in¯orescences using primers that identi®ed either the endogenous FIE or the GFP:FIE transcripts. Ampli®cation of ubiquitin (UBI) was used as internal control. (b) Detection of FIE by Western analysis of protein extracts from wt and FIEsilenced rosette leaves using anti-FIE antibodies (left panel). Equivalent amounts of protein extracts were loaded as determined by staining the membrane with Ponceau-red (right panel). (Figure 2a,b). Occasionally, stems were fasciated (Figure 2e,f), indicating enlargement of the shoot apical meristem. Whereas FIE-silenced and wild-type plants developed the same number of internodes along the in¯orescence stem, in FIE-silenced plants the internodes were shorter, resulting in stunted plants (Figure 2b). Under long photoperiod (16 h light/8 h dark), FIE-silenced plants ¯owered, on average, 21 days after germination, exhibiting eight rosette leaves, while wild-type plants ¯owered, on average, 27 days after germination, producing 11±13 rosette leaves. These results support earlier ®ndings indicating that FIE suppresses ¯owering during the late stages of embryogenesis (Kinoshita et al., 2001), revealing a similar function of FIE during the vegetative phase of development. The ®rst four to six rosette leaves of FIE-silenced plants (part of the vegetative phase) exhibited normal morphology, while the successive rosette leaves were narrower than the wild type, rolled up towards the midrib and displayed varying degrees of serration (Figure 2c,h). Occasionally, stipules were longer than those of wild-type plants, and developed stigmatic-like ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 709 papillae at the tip (Figure 2g). Cauline leaves were smaller than those of wild-type plants, and their margins rolled up towards the midrib (Figure 2i,j). Occasionally, stigmaticlike papillae and initials of ovule-like primordia developed at the marginal tips of cauline leaves (Figure 2j), indicating a homeotic conversion to carpel-like organs. The in¯orescences produced fewer ¯owers than those in wild-type plants, and often developed terminal ¯owers in which the outer whorls (sepals and petals) formed carpeloid-like organs (Figure 2o). Stem fasciations caused ¯ower clustering and loss of normal phylotaxy (Figure 2e,f). Flower organ abnormalities were observed in all four whorls (Figure 2k±s). The ®rst whorl had narrow sepals, white at their tips and occasionally serrated (Figure 2p). The second whorl had narrower petals (Figure 2q), often emerging between unopened sepals (data not shown). Frequently, stamenoid petals (Figure 2q) and petaloid stamens (Figure 2r) developed, resulting in variable numbers of stamens, ranging from ®ve to seven. In addition, stamens often produced a reduced amount of pollen. Some stamens developed stigmatic-like papillae at their tips (Figure 2o). In most ¯owers of the FIE-silenced plants, the ovaries were misshapen, comprising three fused carpels (Figure 2s). Occasionally, several carpels were fused to form a complex ¯ower (Figure 2f). FIE-silenced ovules often had a longer funiculus, and some developed stigmatic papillae at their tips and subtending ovules at their margins, indicating a homeotic transformation of the ovules into a carpel-like structure (Figure 3). The fertility of FIE-silenced ¯owers was low, and ¯oral organs tend not to detach after anthesis (Figure 2b). Plant senescence was also found delayed, possibly because of a reduction in fertilization, as observed in various male sterile mutants (Chaudhury et al., 1994). Thus, the reduction in FIE levels (Figure 1) interfered with a wide range of developmental processes, demonstrating the central role of FIE as an important regulator during plant vegetative and reproductive development. KNOX genes are de-repressed in FIE-silenced plants In Drosophila, mutations in the ESC gene result in homeotic conversions and ectopic expression of transcription factors. Similarly, in FIE-silenced plants it is plausible that the observed homeotic changes result from the ectopic expression of meristem and organ identity transcription factors. The serrated leaf phenotype observed in FIE-silenced plants suggested that FIE participates in the regulation of BREVIPEDICELLUS (BP), a member of class I KNOX genes (Ori et al., 2000). Expression of all class I KNOX genes was examined by RT-PCR, and showed that levels of BP, KNAT2 and SHOOTMERISTEMLESS (STM) (Figure 4) were signi®cantly higher in FIE-silenced plants than in wild-type plants. No signi®cant differences in the expression of KNAT6 were observed (Figure 4). 710 Aviva Katz et al. Figure 2. Abnormal phenotypes displayed by FIE-silenced plants. (a, b) Wild-type (wt) and FIE-silenced adult plants, respectively. (c, d) Close-up of FIE-silenced and wt rosette leaves, respectively. (e, f) Fasciated stem and multi-carpel ¯owers in FIE-silenced plants, respectively. (g) Abnormal elongated stipules with stigmatic-like papillae structures at the tip. (h) wt and FIE-silenced rosette leaves. Note the serration, size and curling of leaves. (i, j) Cauline leaves of wt and FIE-silenced plants. (k±o) wt (k) and different ¯owers of FIE-silenced plants (l±o). Note the carpeloid organ with stigmatic-like papillae in the ®rst whorl (o), the stamenoid petals (m) and the multi-carpel gynoecia (n). (p±s) Variable phenotypes of four different ¯ower organs: sepals, petals, stamens and carpels, respectively. Left panels show wt organs. Note the bifurcated petal and petaloid sepal (p), the stamenoid petals (q), the petaloid stamens (r) and the multi-carpel gynoecia (s). De-repression of BP was con®rmed by examining BP:bglucuronidase (GUS) expression patterns in the FIEsilenced background. Plants carrying BP promoter fused to GUS reporter gene were crossed with FIE-silenced plants, and the expression patterns of the reporter were examined (Figure 5). In wild-type plants, the GUS expression pattern was similar to that reported by Ori et al. (2000), i.e. it was found in the veins of cotyledons, in shoot meristem and in the basal part of veins (Figure 5a,c). We also detected expression in the hydathodes of rosette leaves (Figure 5c). In the FIE-silenced background, GUS expression extended to the vasculature of rosette leaves (Figure 5b,d). The abnormal GUS staining could be detected in both the ®rst four normal-looking rosette leaves and in successive leaves with abnormal morphology, indicating that the severity of FIE-silenced leaf morphology may depend on the expression of additional genes. ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 FIE regulates homeobox gene expression in sporophyte development Figure 3. Homeotic transformation of FIE-silenced ovules into carpel-like structures. (a) Ovary with a protruding spaghetti-like ovule; Insert, close-up of carpellike ovule with stigmatic papillae. (b) Nomarsky differential interference contrast image of a carpel-like ovule with secondary ovules (see insert). Scale bars are 0.2 mm. ASYMMETRIC LEAVES1 (AS1) and AS2 are known to negatively regulate class I KNOX genes, including BP and KNAT2 (Byrne et al., 2002; Ori et al., 2000; Semiarti et al., 2001). To discover whether FIE controls class I KNOX genes via the regulation of AS1 and AS2, the steady-state RNA levels of AS1 (Figure 4) and AS2 (data not shown) were tested in rosette leaves and found to be unchanged, suggesting that AS1 and AS2 are not likely to function downstream to FIE. FIE represses expression of homeotic genes RT-PCR analysis of wild-type and abnormal rosette leaves (Figure 4), cauline leaves and sepals (see Supplementary Material, Figure S1) revealed that transcript levels of three MADS-box genes, AGAMOUS (AG), APETALA3 (AP3) and 711 Figure 5. Expression of pBP:GUS in FIE-silenced plants. (a) An 18-day-old wild-type seedling expressing pBP:GUS. (b) An 18-day-old FIE-silenced seedling ¯owering prematurely. (c) A detached rosette pBP:GUS wild-type leaf, exhibiting GUS expression in the basal part of the veins and in the hydathodes. (d) Detached rosette leaves from FIE-silenced plant expressing pBP:GUS. The GUS staining is apparent in the vasculature of the fourth rosette leaf displaying a normal phenotype and in a younger leaf, which is small, curled and serrated, characteristic of abnormal FIE-silenced plants. AGAMOUS LIKE17 (AGL17), as well as a PcG SET domain protein MEA were signi®cantly elevated (Figure 4). The steady-state RNA levels of PISTELLATA (PI) and AP1 were not affected. No changes were detected in steady-state transcript levels of known AG regulators such as WUSCHEL (WUS) (Lenhard et al., 2001; Lohmann et al., 2001), AINTEGUMENTA (ANT ) (Krizek et al., 2000) and APETALA2 (AP2) (Bowman et al., 1991; Drews et al., 1991; Kunst et al., 1989; Modrusan et al., 1994). The expression level of SUPERMAN (SUP), a regulator of AP3 (Bowman et al., 1992; Sakai et al., 1995), was also not altered. Data for most of the genes that showed no change in transcript levels are not shown. Ectopic expression of AG in FIE-silenced plants promotes malformation of cauline leaves Figure 4. Expression analysis of FIE-silenced rosette leaves. RT-PCR analysis detecting transcripts of: endogenous FIE; homeotic genes AG, AGL17 and AP3; SET-domain proteins MEA and CLF; homeobox genes BP, KNAT2, STM, KNAT6; and AS1. Ampli®cation of rRNA transcripts was used as internal control. The number of ampli®cation cycles was optimized for each cDNA to allow detection in the exponential range of ampli®cation as visualized by ethidium bromide staining of the samples separated on agarose gel electrophoresis. ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 It has been reported that the ectopic expression of AG induces the development of small and curled cauline leaves (Goodrich et al., 1997; Mizukami and Ma, 1992). Thus, the malformed cauline leaves typical of FIE-silenced plants may be the result of de-repression of AG. To test this hypothesis, we generated ag-1 / /FIE-silenced mutant plants. To this end, crosses were carried out between independent FIEsilenced lines and ag-1 heterozygote plants. ag-1 / /FIEsilenced F2 plants were identi®ed and con®rmed by RT-PCR to be co-suppressed for the expression of both endogenous FIE and transgenic GFP:FIE (data not shown). Unlike the FIEsilenced phenotype, in the absence of a functional AG, ag-1 / /FIE-silenced plants displayed rosette and cauline leaves, which were not curled (Figure 6). These results indicate that AG overexpression indeed contributed to 712 Aviva Katz et al. Figure 7. Similarities of gene expression pattern in rosette leaves of FIEsilenced plants and clf-2 mutant. Total RNA was extracted from rosette leaves of Arabidopsis wild-type (wt) Landsberg erecta (La(er)), Columbia (Col), clf-2 and FIE-silenced plants. RNA was ampli®ed by RT-PCR using primers identifying the transcripts of AGL17, AG, AP3, BP, KNAT2, KNAT6, STM, AS1 and MEA. Products were resolved by agarose gel electrophoresis and stained with ethidium bromide. rRNA ampli®cation served as an internal control. Figure 6. Phenotypes of leaves and ¯owers of ag-1/FIE-silenced plants. Rosette, cauline, sepals and petal leaves from wild-type Landsberg erecta La(er), ag-1 / , FIE-silenced and ag-1 / /FIE-silenced plants are shown. Representing samples were taken from plants of the same developmental stage. the curled leaf phenotype of FIE-silenced plants. As in ag-1 / /FIE-silenced plants, the ¯owers lacked stamens and carpels, it was not possible to determine whether AG has a role in the development of aberrant carpel and ovule phenotypes. FIE and CLF control common developmental programmes FIE-silenced and clf mutant plants showed similar morphology of rosette and cauline leaves, ¯owers, length of in¯orescence stem internodes, ¯owering time and ectopic expression of AG and AP3 (Goodrich et al., 1997). RT-PCR analysis revealed that CLF expression was not altered in the rosette leaves of FIE-silenced plants (Figure 4), indicating that the upregulation of affected genes did not result from the downregulation of CLF. To ®nd out whether FIE and CLF regulate the same set of genes, we compared the expression levels of the identi®ed derepressed genes in FIE-silenced plants and clf mutants. As shown in Figure 7, in the rosette leaves of both FIEsilenced plants and clf-2 mutant, the expression of AGL17, AG, AP3, KNAT2, STM and MEA was upregulated. The above results, together with the fact that CLF is a SET domain PcG protein, strongly suggested that CLF and FIE share common function, suppressing expression of key developmental transcriptional regulators. BP expression was upregulated only in FIE-silenced rosette leaves, but not in clf-2, indicating that FIE may act via the association of proteins other than CLF to control expression. Interaction between FIE and CLF proteins The common morphological characteristics of clf and FIEsilenced plants, the fact that these proteins regulate a common set of genes, and the homology of FIE and CLF ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 FIE regulates homeobox gene expression in sporophyte development 713 Figure 8. FIE and CLF interact in yeast and in vitro. (a±c) Yeast two-hybrid assays with cells expressing: GAL4BD:FIE (FIE); GAL4AD:CLF (CLF); GAL4BD:FIE with GAL4BD:CLF (FIE CLF); and GAL4BD:FIE with GAL4AD:MEA (FIE MEA). Cells were grown on supplemented minimal media lacking leucine and tryptophan ( LT) (a) or leucine, tryptophan and histidine ( LTH) (b). Colonies grown on plate (b) were lifted on ®lters and assayed for b-galactosidase activity (c). Growth on media lacking histidine (b) and blue colour (c) indicates interaction between FIE and either MEA (as a positive control) or CLF. (d) Autoradiogram displaying radioactively labelled FIE that was pulled down by increasing concentrations of immobilized GST:CLF (triangle). In vitro transcribed and translated 35S-methionine-labelled FIE (Input) was incubated with immobilized GST (20 mg) or GST-CLF protein (5, 10 and 20 mg), washed, eluted and subjected to SDS±PAGE and autoradiography. The input FIE (Input) represents 10% of the amount of 35S-methionine-labelled FIE incubated with immobilized proteins. (e) Immunodetection of the in vitro transcribed FIE by polyclonal FIE antibody. to known PcG WD40 and SET domain proteins, which interact in Drosophila, mouse and humans (reviewed by Berger and Gaudin, 2003; Simon and Tamkun, 2002), suggest that FIE and CLF may function together in a regulatory complex to suppress gene expression. To test whether FIE and CLF interact, we used yeast twohybrid assays (Figure 8). Yeast expressing either GAL4BDFIE or GAL4AD-CLF did not self-activate expression of the reporter genes. Yeast expressing both GAL4BD-FIE and GAL4AD-AtHD2a (Wu et al., 2000), serving as a negative control, did not activate expression of the reporter gene (data not shown). However, yeast expressing both GAL4BD-FIE and GAL4AD-CLF was found to activate the HIS3 (Figure 8b) and b-galactosidase (Figure 8c), indicating that FIE interacts with CLF. An in vitro pull-down assay was performed to validate the results of the yeast two-hybrid assays (Figure 8). To this end, CLF was expressed fused to glutathione S-transferase (GST:CLF), and tested for its ability to bind radioactively labelled FIE. The identity of the labelled fulllength FIE was con®rmed by its apparent size of 41 kDa (Figure 8d), and by an anti-FIE antibody (Yadegari et al., 2000; Figure 8e). Radioactively labelled FIE was retained by the immobilized GST:CLF, but not by immobilized GST alone (Figure 8d). Moreover, the quantity of retained labelled FIE correlated with the amount of immobilized GST:CLF used (Figure 8d), indicating the speci®city of the interaction. Taken together, the morphological, molecular and interaction assays strongly suggest that FIE and CLF interact in planta to form a PcG complex, which regulates sporophyte development. ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 Discussion FIE controls sporophyte development The FIE-silenced plants generated in the framework of this study developed through their vegetative and reproductive phases, to reach maturity, thus allowing us to study FIE function and identify FIE-regulated genes and potential partners for interaction with FIE. FIE-silenced plants displayed a variety of morphologically abnormal phenotypes, which correlated with reduced levels of FIE transcript and protein (Figure 1). We postulate that co-suppression occurred subsequent to germination, allowing FIE-silenced plants to develop beyond the seedling stage. This assumption is supported by the ®nding that emasculated FIE-silenced plants neither developed autonomous endosperm or seed-like structures, nor did they abort the embryo when fertilization took place (data not shown), a characteristic of ®e mutants (Ohad et al., 1996). Although low levels of FIE transcripts were detected in abnormal FIE-silenced plants (Figure 1), these levels were probably not suf®cient to allow for normal development. ESC, the Drosophila homologue of FIE, forms a large multiprotein PcG complex, which is highly sensitive to the initial concentrations of its components, leading to an all-or-none complex formation (Pirrotta and Rastelli, 1994). Similarly, FIE is likely to be part of a multiprotein complex (Kohler et al., 2003a; Luo et al., 1999, 2000; Spillane et al., 2000; Yadegari et al., 2000; Figure 8). Thus, we assume that the low levels of FIE protein in FIE-silenced plants limited the 714 Aviva Katz et al. formation of FIE±PcG complex, resulting in the observed pleiotropic phenotypes. FIE is required to maintain the repression of homeobox genes PcG proteins maintain the repressed states of homeotic gene expression during development (Simon and Tamkun, 2002). In Drosophila and mammals, mutations in PcG proteins lead to de-repression of their target genes, resulting in altered developmental programmes, including homeotic transformations. In Arabidopsis, the reduction of FIE levels induced de-repression of key regulatory genes that control apical dominance, ¯owering time, and leaf ¯ower and ovule development, resulting in homeotic transformations (Figures 4, 5 and 7). The plant PcG proteins FIE, MEA, CLF, VRN2 and EMF2 were shown to regulate MADS-box gene expression (Gendall et al., 2001; Goodrich et al., 1997; Kinoshita et al., 2001; Kohler et al., 2003b), whereas in Drosophila, the PcG complexes were found to regulate homeobox genes. Here, we present evidence that the PcG proteins FIE and CLF play a major role in maintaining the repression of homeobox gene outside their appropriate temporal and spatial expression boundaries. This may imply that the regulatory mechanism involving the control of homeobox genes by PcG complexes developed prior to the evolutionary divergence of the plant and animal kingdoms, and that its functional role was conserved. FIE-regulated homeobox genes BP, KNAT2 and STM (Figures 4, 5 and 7), are members of the class I KNOX genes, known to regulate shoot meristem maintenance (Lincoln et al., 1994; Long et al., 1996; Reiser et al., 2000). Downregulation of KNOX genes during the development of leaf primordium is required for normal leaf morphogenesis and persists in the mature leaf (Dockx et al., 1995; Lincoln et al., 1994; Long et al., 1996; Semiarti et al., 2001). The ectopic expression of BP:GUS in an FIE-silenced background (Figure 5) demonstrates that FIE controls the expression boundaries of BP. De-repression of BP, KNAT2 and STM correlates with the FIE-silenced phenotype, mimicking some of the phenotypes described for plants overexpressing class I KNOX genes. STM overexpression induces the formation of ectopic meristems (Brand et al., 2002), and is associated with abnormal leaf development (Brand et al., 2002; Gallois et al., 2002; Lenhard et al., 2002). The phenotypes of FIE-silenced leaves resemble those observed in plants overexpressing BP, resulting in lobed leaves reduced in size and the formation of ectopic meristems on leaves (Chuck et al., 1996; Reiser et al., 2000). KNAT2 overexpression causes curling of leaves, induces stigmatic papillae on rosette leaves and converts ovules into carpel-like structures (Pautot et al., 2001), resembling the phenotypic abnormalities observed in FIE-silenced plants. Thus, in FIE-silenced plants, ectopic expression of BP may have contributed to serration of the leaves (Figure 2), while the ectopic expression of KNAT2 may contribute to leaf curl and the homeotic conversion of ovules into carpels (Figure 3). Abnormal carpel development (Figure 2) is also consistent with the ectopic expression of AG (Figure 4), a known regulator of carpel development. Carpel development is also regulated by AP2 (Bowman et al., 1991) and BELL1 (BEL1) (Modrusan et al., 1994; Robinson-Beers et al., 1992). Mutations in these genes cause the transformation of ovules into carpel-like structures. However, there was no change in expression levels of BEL1 and AP2 in leaves of FIE-silenced plants compared with the wild type (data not shown). This shows that the conversion of ovules into carpel-like organs may have been induced directly by KNAT2, or by other genes yet to be determined. Putative candidates are SEPALLATA1 (SEP1), SEP2, SEP3, SHATTERPROOF1 (SHP1) and SHP2, which are known effectors of carpel development (Favaro et al., 2003; Pelaz et al., 2000). AS1 and AS2 were found to suppress class I KNOX genes in leaves (Byrne et al., 2000; Semiarti et al., 2001). As AS1 (Figure 4) and AS2 (data not shown) transcript levels remained unchanged in the leaves of FIE-silenced plants, it is likely that FIE represses class I KNOX genes through a different pathway. This hypothesis is supported by the different gene expression pro®le exhibited by FIE-silenced plants as compared with the reported pro®les of as1 and as2 mutants. Whereas BP, KNAT2 and KNAT6 are expressed in as1 and as2 leaves (Byrne et al., 2000; Semiarti et al., 2001), FIE-silenced plants express BP, KNAT2 and STM (Figure 4). Furthermore, as1 and as2 mutants have been shown to exhibit asymmetric leaf lobes and downward curling of leaves, and thus are phenotypically distinct from leaves of FIE-silenced plants. Ectopic expression of BP, KNAT2 and STM has been shown in leaves of the double mutants ®l yab3-2 (Kumaran et al., 2002). However, the expression of FILAMENTOUS FLOWER (FIL) and YABBY3 (YAB3; data not shown) appears to be unchanged in FIE-silenced plants, implying that FIE may repress the expression of KNOX genes using different pathways. The homeotic conversion of leaves and ¯ower organs into carpeloid structures and the molecular evidence provided by this study indicate that another group of FIEregulated genes, including AG and AP3, belongs to the MADS-box gene family, which is consistent with the ®ndings of other studies (Yadegari et al., 2000). The ectopic expression of homeobox and MADS-box genes described in this work establishes that in wild-type plants, FIEmediated repression occurs throughout the entire vegetative phase of plant development. The C-class organ identity gene, AG, is known to regulate stamen and carpel development (Bowman et al., 1989; Yanofsky et al., 1990), as well as ¯oral meristem determinacy (Busch et al., 1999; Mizukami and Ma, 1995; Okamuro ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 FIE regulates homeobox gene expression in sporophyte development et al., 1996). FIE-silenced plants, which ectopically expressed AG, resembled partially AG-overexpressing plants (Figures 2, 4 and 6). Accordingly, the curled leaf phenotype of FIE-silenced plants was suppressed in the ag-1 background (Figure 6). These results demonstrate that AG overexpression plays a role in the curled leaf phenotype. KNAT2 was ectopically expressed in FIE-silenced rosette leaves (Figure 4). Ectopic expression of KNAT2 was shown to induce expression of AG (Pautot et al., 2001). No changes in the expression levels of other AG regulators, such as WUS (Lenhard et al., 2001), AP2 (Bowman et al., 1991; Drews et al., 1991; Kunst et al., 1989; Modrusan et al., 1994), or ANT (Krizek et al., 2000) were detected (data not shown). Thus, AG-elevated expression either may have resulted from the direct de-repression of AG in the absence of the FIE±PcG complex, or may have been activated by KNAT2. Similarly, CLF may regulate AG expression via KNAT2, as indicated from the upregulation of KNAT2 in clf mutants (Figure 7). It is interesting to note the phenotypic resemblance between FIE-silenced elongated ovules and those observed in petunia, which resulted from co-suppression of Fbp7 and Fbp11 MADS box-like genes that are regulators of ovule identity (Angenent et al., 1995). However, in FIE-silenced plants, we detect no change in expression level of SEEDSTICK (STK; previously AGL11), which is considered to be the Arabidopsis orthologue of Fbp7 and Fbp11 (Becker and Theissen, 2003). Other members of the MADS-box gene family, AG and AGL17, were upregulated (Figures 4 and 7). As in wild-type plants AGL17 expression is restricted to the roots (Burgeff et al., 2002; Rounsley et al., 1995), the importance of FIE repression of AGL17 expression in the shoot remains to be determined. FIE may form alternative functional complexes with different PcG SET domain proteins FIE and CLF interaction, demonstrated by yeast two-hybrid and in vitro binding assays (Figure 8), implies that both proteins may interact in vivo to form a PcG complex. Moreover, the leaf and ¯ower phenotypes of FIE-silenced plants resemble those of clf mutants (Goodrich et al., 1997), while both proteins regulate a common set of transcription factors, members of the homeobox and MADS-box gene families (Figure 7). Thus, FIE interacts with different members of the SET domain protein family such as MEA (Luo et al., 1999, 2000; Spillane et al., 2000; Yadegari et al., 2000) and CLF (Figure 8), possibly allowing the formation of various PcG complexes, which regulate different developmental programmes. Supporting the above is the observation that BP is de-repressed in FIE-silenced plants but not in clf-2 mutants, indicating that FIE may associate with different PcG proteins other than CLF, in order to mediate represß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 715 sion of BP. The dynamics of PcG's organization during developmental programmes has been shown in Drosophila (Furuyama et al., 2003). De-repression of MEA was observed in rosette leaves of both clf and FIE-silenced plants (Figures 4 and 7). These results indicate that in wild-type plants, the PcG complex containing both FIE and CLF downregulates MEA expression in the sporophyte. Such regulation of MEA expression may lead to the formation of a FIE±CLF complex and suppress the formation of FIE±MEA complex in the sporophyte. DEMETER (DME), another known regulator of MEA, causes ectopic expression of MEA in rosette leaves when overexpressed (Choi et al., 2002). However, it appears that DME did not mediate the upregulation of MEA in FIEsilenced plants and clf-2 mutants because DME expression levels were not changed in these plants (data not shown). Potential partners of Arabidopsis PcG complexes FIE PcG complexes maintain the restricted pattern of various homeobox and MADS-box gene expression, thus mediating the control of reproductive and vegetative programmes. The results of this study outline the unique ability of FIE in plants to interact with different PcG SET domain proteins. Genetic and biochemical studies may help to determine potential constituents of these complexes. The Drosophila ESC±Ez complex was shown to contain p55, an Rb-binding protein, and Su(z)12, a zinc ®nger protein (Luo et al., 1999; Muller et al., 2002; Ng et al., 2000; Tie et al., 2001). In Arabidopsis, recent ®ndings show that the FIE±MEA complex in seeds contains MSI (Kohler et al., 2003a), a homologue of p55 (Ach et al., 1997), which extends the structural conservation between the plant and Drosophila's PcG complexes. FIS2, a zinc ®nger protein, was suggested to be part of this complex because of the similarities between the mutant phenotypes of ®s2 and those of ®e, mea and msi1, initiating autonomous endosperm development. FIE, MEA and FIS2 repress the expression of PHE, which further supports their participation in a common PcG complex (Kohler et al., 2003b). The proposed FIE±CLF complex may also harbour EMF2, a homologue of Su(z)12, as a zinc ®nger member of the complex. This model is supported by phenotypic similarities among the various mutants. For example, ®e (Kinoshita et al., 2001), clf (Goodrich et al., 1997) and emf2 (Sung et al., 1992) mutants all display early ¯owering, and clf (Goodrich et al., 1997), co-suppressed FIE and co-suppressed EMF2 (Goodrich et al., 1997) display the curled leaf phenotype. Genetic analysis (Haung and Yang, 1998) reveals that EMF2 independently controls ¯owering time and rosette leaf formation, thus further supporting the above notion that EMF2 may function together with FIE and CLF to regulate the vegetative phase. The FIE±CLF±EMF2 complex proposed here possibly contains MSI1, similar to the putative 716 Aviva Katz et al. FIE±MEA±FIS2±MSI1 complex in seeds, just as the MSI1 homologue was found to be part of the Drosophila PcG complex. This is further supported by the similarity in phenotype of FIE and MSI co-suppressed plants (Hennig et al., 2003), displaying reduced apical dominance, curled leaves, and aberrant ¯owers and ovules. Experimental procedures Plant material and growth conditions Arabidopsis thaliana ecotype Columbia glabra served as the wild type. clf-2 and ag-1 mutants were obtained from the Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA). Plants were grown at 228C under 16-h light and 8-h dark photoperiods in Percivall growth chambers. Plant morphology Plant morphology was documented using a SV11-stereomicroscope and Axioplan-2 microscope (Carl Zeiss Inc., Gottingen, Germany) and photographed with a Nikon-coolpix 950 (Tokyo, Japan) camera. Image processing was performed with PHOTOSHOP 7.0.2 (Adobe Systems, San Jose, CA, USA). Generation of transgenic plants p35S-GFP:FIE was constructed as a translational fusion between GFP(S65T) and FIE cDNA at N-terminus. First, FIE cDNA was inserted between the CaMV-35S promoter and the polyadenylation sequence, using SalI and XmaI sites of pMD1 binary vector to create pMDI-FIE. pMD1 is a derivative of the pBI121 vector (Clontech, Palo Alto, CA, USA) in which the GUS gene was replaced by a multiple cloning site. In the second stage, the GFP was ampli®ed by PCR, with a forward primer harbouring both BamHI and NcoI sites (50 -GCGGATCCATGGTGAGC-30 ), and a reverse primer, which abolished the stop codon and contained a BamHI site (50 GCGGATCCCTTGTACAGCTCGTCC-30 ). The PCR product was digested with BamHI and subcloned into the pMDI-FIE vector, resulting in the pMD1-GFP:FIE. A 15-bp spacer encoding a KGSPG amino acid sequence was added between the FIE- and GFP-coding sequences because of the cloning procedure. All constructs were veri®ed by sequencing. The binary vector was transformed into Agrobacterium GV3101 pMP90 (Koncz and Schell, 1986). Transformation of Arabidopsis was performed by the ¯oral dip method (Clough and Bent, 1998). Sixty independent kanamycin-resistant T1 plants were obtained and con®rmed to contain the desired insert by PCR. Total RNA isolation and RT-PCR analyses Sample tissue was collected from Arabidopsis plants and immediately frozen in liquid nitrogen. Total RNA was prepared using an SV total RNA isolation kit (Promega, Madison, WI, USA). For reverse transcription, 12 mg of total RNA was incubated for 1 h at 428C with 300 units of Moloney Murine leukaemia virus H superscript reverse transcriptase (Invitrogen Corp., Carlsbad, CA, USA) in 70 ml reaction mixture containing 215 pmol of 21oligo (dT), appropriate buffer, 10 mM DTT, 0.6 mM dNTPs (Roche, Mannheim, Germany) and primer 3404 (50 -ACATCTAAGGGCAT- CACAGAC-30 ) for priming ribosomal RNAs. cDNAs equalled to one-seventh of the total RNA in the reverse transcription reaction mixtures were ampli®ed with ExTaq polymerase (Takara, Otsu, Japan) in 50 ml of reaction mixtures. 18S ribosomal RNA served as an internal control for monitoring reaction ef®ciency and to assure that the initial amounts of cDNA were equal. 18S ribosomal RNA was ampli®ed with (50 -TGCAGTTAAAAAGCTCGTAGTTG-30 ) and (50 -ACATCTAAGGGCATCACAGAC-30 ) primers. The GFP:FIE transgene was ampli®ed with a GFP forward primer (50 -GCGGATCCATGGTGAGCAAGG-30 ) and an FIE reverse primer (50 -CCGCTCGAGCTACTTGGTAATCACGTC-30 ). Endogenous FIE cDNA was ampli®ed with a forward primer (50 -GATTGTCGACTCGAGATGTCGAAGATAACC-30 ) and a reverse primer from the 30 untranslated region (50 -CTCCAGAAAGGGTATACACTG-30 ). Expression of MADS-box and homeobox genes was examined using a self-developed RTPCR ampli®cation kit (Patent case # 133684, Tel Aviv, Israel). Other speci®c cDNA targets were ampli®ed using the following primer sets: MEA (50 -GCAGGACTATGGTTTGGATG-30 ) and (50 GATCAGAGGATTGGTCTATTTGC-30 ); AP3 (50 -ATGGCGAGAGGGAAGATCCAG-30 ) and (50 -GATGGCACCAGCAAACCTTTTAG-30 ); AGL17 (50 CGGGATCCTAGAACGCACCAAGATCTAAAGG-30 ) and (50 -CGGCTCGAGTTAGCTGTTTGAAGATGTCTTATAATGG-30 ); CLF (50 -ATGGCGTCAGAAGCTTCGC-30 ) and (50 -CTTCCAGACTTGAGAAGCG-30 ); AG (50 -CTAGGAGGAGATTCCTCTCC-30 ) and (50 -CTAACTGGAGAGCGGTTTGG-30 ); STM (50 -GTGCTCCTGCCTATTCTCTAATG-30 ) and (50 -CTATCCTCAGTTGTGGATCTAC-30 ); BP (50 -GGGTATGGAAGAATACCAGC-30 ) and (50 -TATGGACCGAGACGATAAG-30 ); KNAT2 (50 -GAAGAGATTCAGCGAGAGAACC-30 ) and (50 -GAATCGTCCATCATATCAAACGGCATG-30 ); KNAT6 (50 -GATGATGTCACCGGAGAGTCTC-30 ) and (50 -GACTCGACACCAGTACATAGGTTC-30 ); AS1 (50 ATGAAAGAGAGACAACGTTGGAG-30 ) and (50 -GAACACACTCTCGCTACTC-30 ); and AS2 (50 -CTCTCAATTTTCAATGGCGGCTTTGTG-30 ) and (50 -CTCAAGACGGATCAACAGTACG-30 ). The annealing temperature used for all sets of primers was 608C. Yeast two-hybrid clones and assays Full-length FIE cDNA was subcloned into the pBI880 vector containing the GAL4-binding domain. The full-length CLF cDNA was ampli®ed by PCR and subcloned into the pBI771 vector containing the GAL4 activation domain (Kohalmi et al., 1997). All clones were veri®ed by sequencing. Yeast two-hybrid assays were carried out as previously described by Yadegari et al. (2000). Detection of protein±protein interaction by pull-down assay Pull-down assays were performed according to Yadegari et al. (2000). 35S-radioactively labelled FIE was produced as previously described by Ohad et al. (1999), by expressing FIE cDNA in the pCITE vector (Novagen, Darmstadt, Germany), allowing coupled transcription±translation of the FIE protein radioactively labelled by 35S-methionine. CLF full-length cDNA was cloned by RT-PCR, and subsequently subcloned into the pGEX-4T-1 vector (Amersham Pharmacia Biotechnology, Piscataway, NJ, USA), allowing expression and puri®cation of the CLF-GST fusion protein. Puri®ed GST:CLF (5, 10 and 20 mg) or control GST (20 mg) proteins were immobilized on glutathione-agarose beads (Sigma, St Louis, MO, USA), washed with 20 mM Tris±HCl buffer (pH 8.0) containing 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet-P40, and re-suspended in 200 ml of the same buffer to which the labelled FIE was added and incubated for 2 h at 48C. The beads were washed four times with the same buffer, re-suspended in sample buffer ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719 FIE regulates homeobox gene expression in sporophyte development and resolved by SDS±PAGE. The proteins were transferred to polyvinylidene ¯uoride (PVDF) membranes (Millipore, Bedford, MA, USA). Labelled proteins were detected by autoradiography after exposing the membranes to X-ray BIOMAX MS ®lm (Kodak, Rochester, NY, USA). Total Arabidopsis protein extraction and Western blotting Rosette leaves were harvested and ground to powder in liquid nitrogen. The tissue was further ground with protein extraction buffer (100 mM Tris (pH 7.2), 10% sucrose, 5 mM MgCl2, 5 mM EDTA, 40 mM b-mercaptoethanol and protease inhibitor cocktail (Roche, Mannheim, Germany) in a 1 : 1 ratio to the tissue weight. The extract was then centrifuged, and the supernatant was used for Western blot analysis. Total protein concentration was determined by Bradford assay (Bradford, 1976), and equal amounts of protein were resolved on a 10% SDS±PAGE and transferred onto nitrocellulose membranes. To detect FIE protein, membranes were incubated with rabbit anti-FIE polyclonal antibody (Yadegari et al., 2000) in a 1 : 500 dilution, and then washed and incubated with blotting-grade horseradish peroxidase-conjugated goat-antirabbit secondary antibody (Jackson Immuno Research, West Grove, PN, USA). Detection was carried out using paracoumaric acid and luminol (Sigma, St Louis, MO, USA), according to the manufacturer's instructions. Acknowledgements We wish to thank Dr Ben-Tzion Vider for his assistance in developing a PCR-based kit to examine the expression of gene families; Dr Naomi Ori for providing the BP:GUS seeds, The Arabidopsis Biological Resource Center for providing ag-1 and clf-2 seeds; and Daniella Bar-El, Marina Kalis and Noa Zecharia for technical assistance. We thank Dr S. Yalovsky and Dr N. Ori for their critical reading of the manuscript. O.H. was supported by the Ministry of Science Eshkol Fellowship, Israel. This research was supported by Israel Science Foundation Grant No-503-00, and by BARD, the United States±Israel Binational Agricultural Research and Development Fund, Grant No. IS-3158-99C. Supplementary Material The following material is available from http://www.blackwell publishing.com/products/journals/suppmat/TPJ/TPJ1996/TPJ1996sm. htm Figure S1. Expression analysis of different FIE-silenced plants tissues. Ethidium bromide-stained gels detecting FIE, AG, AGL17, MEA, AP3 and BP transcripts by RT-PCR analysis. Total RNA was extracted from rosette leaves, cauline leaves and sepals of wild-type and FIE-silenced plants exhibiting abnormal phenotypes. Ampli®cation of ubiquitin (UBI) transcripts was used as internal controls. References Ach, R.A., Taranto, P. and Gruissem, W. (1997) A conserved family of WD-40 proteins binds to the retinoblastoma protein in both plants and animals. Plant Cell, 9, 1595±1606. 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