Upon hormonal signaling, ovules develop as lateral organs from the placenta. Ovule numbers ultimately determine the number of seeds that develop, and thereby contribute to the final seed yield in crop plants. We demonstrate here that CUP-SHAPED COTYLEDON 1 (CUC1), CUC2 and AINTEGUMENTA (ANT) have additive effects on ovule primordia formation. We show that expression of the CUC1 and CUC2 genes is required to redundantly regulate expression of PINFORMED1 (PIN1), which in turn is required for ovule primordia formation. Furthermore, our results suggest that the auxin response factor MONOPTEROS (MP/ARF5) may directly bind ANT, CUC1 and CUC2 and promote their transcription. Based on our findings, we propose an integrative model to describe the molecular mechanisms of the early stages of ovule development.
In Arabidopsis, ovules arise from the placenta as lateral organs, and both auxin synthesis and transport play important roles in their formation (Benková et al., 2003). In the yucca1 yucca4 (yuc1 yuc4) and weak ethylene insensitive8 tryptophan aminotransferase related2 (wei8 tar2) double mutants, the local auxin response is compromised, and the pistil that develops lacks ovules (Cheng et al., 2006; Stepanova et al., 2008). A similar phenotype has been described for mp S319, a MONOPTEROS (MP) partial loss-of-function mutation (Cole et al., 2009; Lohmann et al., 2010). MP encodes a member of the auxin response factor (ARF) family (Hardtke and Berleth, 1998) that binds, as a monomer or as a dimer, to the promoters of target genes to control their transcription (Ulmasov et al., 1997). Although total loss-of-function mp mutants are unable to correctly form embryos, embryo development is unaffected in mp S319 plants, suggesting that the MP dimerization domain is required for activities that mainly occur in the post-embryonic phase of plant development (Lau et al., 2011).
Further indications that auxin may influence the number of ovule primordia were obtained by characterization of the partial loss-of-function mutant pin1-5, which develops pistils that have fewer ovule primordia compared to the wild-type (Bencivenga et al., 2012).
Cytokinins have also been reported to play a role in ovule primordium formation. The cytokinin response1-12 (cre1-12) histidine kinase2-2 (ahk2-2) ahk3-3 triple mutant shows a reduction in the cytokinin response, and the number of ovule primordia is drastically reduced (Higuchi et al., 2004). By contrast, the cytokinin oxidase/dehydrogenase3 (ckx3) ckx5 double mutant, in which cytokinin degradation is affected, develops twice as many ovules as the wild-type (Bartrina et al., 2011). In this regard, it has recently been shown that cytokinin modulates auxin fluxes during ovule development by regulating PIN1 expression (Bencivenga et al., 2012).
In addition to the auxin and cytokinin signaling pathways, transcription factors such as ANT (Elliott et al., 1996), CUC1 and CUC2 (Ishida et al., 2000) have been shown to be important for ovule primordia formation. In the ant single mutant, a reduced number of ovule primordia are formed. Furthermore ant ovules lack the two integuments, and the embryo sac arrests at the one-cell stage, suggesting that ANT has multiple functions during ovule development (Elliott et al., 1996; Klucher et al., 1996; Schneitz et al., 1997; Losa et al., 2010).
Although cuc1 and cuc2 single mutants are very similar to wild-type plants (Aida et al., 1997), cuc1 cuc2 double mutants completely lack the shoot apical meristem, and the two cotyledons are fused to form a cup-shaped structure (Aida et al., 1997). Studying the roles of CUC1 and CUC2 in ovule formation is therefore only possible on adventitious shoots that occasionally develop from cuc1 cuc2 mutant calli in tissue culture (Ishida et al., 2000). In cuc1 cuc2 double mutants, the number of ovules was reduced compared to wild-type, and many of them were found to be sterile (Ishida et al., 2000).
In the present work, we studied CUC1 and CUC2 function during ovule development using RNAi-based silencing of CUC1 under the control of an ovule-specific promoter in the cuc2 mutant background. By crossing these plants with the ant mutant, additive roles for ANT, CUC1 and CUC2 in the determination of ovule numbers were apparent. CUC1 and CUC2 are also involved in the localization and expression of PIN1. In addition, we show that ANT is not expressed in the pistils of the mp S319 mutant, thus confirming that MP controls ANT expression during the reproductive developmental phase. CUC1 and CUC2 were found to be direct targets of MP. Based on our findings, we propose a model for ovule primordia formation in which MP integrates the auxin signaling required for ovule primordium formation to regulate the expression of the transcription factors encoded by ANT, CUC1 and CUC2.
ANT, CUC1 and CUC2 are required for ovule initiation
In the cuc1 cuc2 double mutant and the ant single mutant, ovule numbers are reduced (Elliott et al., 1996; Ishida et al., 2000). To study the interaction between these three genes, CUC1 was silenced in the ant-4 cuc2-1 double mutant background using a CUC1-specific RNAi construct under the control of the ovule-specific SEEDSTICK promoter (pSTK) (Kooiker et al., 2005), which is already active in the placenta before ovule primordia arise (Figure 1a,b).
All mutant combinations were morphologically analyzed by differential interference contrast microscopy. For each genotype, we analyzed ten pistils from each of six plants. The down-regulation of CUC1 due to the specific RNAi was verified using real-time PCR (Figure 1c). The ant-4 (Figure 1e) and cuc2-1 pSTK::CUC1_RNAi mutant plants (Figure 1f) showed reduced ovule number compared to wild-type (Figure 1d), in agreement with previously published data (Elliott et al., 1996; Ishida et al., 2000). The ovules of the ant-4 cuc2-1 double mutant resembled those of the ant-4 single mutant (Figure 1h). In the ant-4 cuc2-1 pSTK::CUC1_RNAi transgenic plants, we observed a further dramatic reduction in the number of developing ovule primordia (Figure 1g). The cuc2-1 pSTK::CUC1_RNAi mutants developed a mean of 30 ovules per pistil, the ant-4 mutant developed a mean of 20 ovules per pistil, and the ant-4 cuc2-1 pSTK::CUC1_RNAi plants developed a mean of seven ovule primordia per gynoecia (Figure 1h), suggesting that ANT, CUC1 and CUC2 act additively in controlling the number of ovule primordia that develop. Despite the reduction in ovule number in the mutant backgrounds, the sizes of the pistils were not reduced (Figure S1). Therefore, the ovules present in the pistils of the mutants are more distantly spaced compared to those of the wild-type (Table S1).
CUC proteins are involved in controlling PIN1 expression and are required for ovule formation
A reduction in the numbers of ovule primordia was also observed in the pin1-5 mutant (Bencivenga et al., 2012) and the cre1-12 ahk2-2 ahk3-3 triple mutant (Higuchi et al., 2004). Recently, it has been proposed that cytokinin regulates PIN1 expression during the early stages of ovule development (Bencivenga et al., 2012), and that PIN1 is required to form ovule primordia (Benková et al., 2003; Bencivenga et al., 2012).
To analyze whether the reduction in ovule number observed in the ant-4 cuc2-1 pSTK::CUC1_RNAi mutant was due to PIN1 down-regulation, we crossed the ant-4/ANT cuc2-1 pSTK::CUC1_RNAi plants with those containing the pPIN1::PIN1-GFP reporter construct (Friml et al., 2003). Analyses of these reporter line plants showed that, during the early stages of pistil development, PIN1–GFP is expressed in the ovule primordia before stage 1-I (Figure 2a) and at stage 1-II (Figure 2b) in the plasma membrane of the epidermal cell layer of the ovule primordia. The expression and localization of PIN1–GFP in the plasma membrane in the ant-4, pSTK::CUC1_RNAi and cuc2-1 ovule primordia were similar to that observed in wild-type plants (Figure S2). In contrast, in the cuc2-1 pSTK::CUC1_RNAi plants, the PIN1–GFP recombinant protein was barely visible and was partially included in vesicles (Figure 2c,d). Furthermore, in cuc2-1 pSTK::CUC1_RNAi plants, PIN1 appears to be expressed in all cells of the primordium as well as in the boundary between ovules (Figure 2d). Real-time PCR analysis showed that PIN1 expression was down-regulated in cuc2-1 pSTK::CUC1_RNAi plants compared to ant-4 single mutants and wild-type plants (Figure 2e). In cuc2-1 and pSTK::CUC1_RNAi mutant plants, the levels of PIN1 expression are not different compared to wild-type (Figure S3). Furthermore, PIN1 down-regulation in ant-4 cuc2-1 pSTK::CUC1_RNAi plants was similar to that in cuc2-1 pSTK::CUC1_RNAi plants (Figure S3). This result suggests that CUC1 and CUC2 may redundantly regulate PIN1 expression during the early stages of ovule development.
To verify whether CUC1 and CUC2 are able to induce PIN1 expression in vivo, we performed transient expression assays in BY-2 tobacco protoplasts. A significant increase in expression of the pPIN1::LUCIFERASE (pPIN1::LUC) reporter, when co-transformed with CUC1 or CUC2, further confirmed that the transcription factors encoded by CUC1 and CUC2 act as positive regulators of PIN1 (Figure 2f). In contrast, PIN7::LUCIFERASE expression was not affected by either CUC1 or CUC2 (Figure 2f).
To understand whether the reduction in ovule number observed in the cuc2-1 pSTK::CUC1_RNAi plants is caused by the down-regulation of PIN1 expression, we up-regulated PIN1 expression in the cuc2-1 pSTK::CUC1_RNAi pistil. It has been shown that treatment with cytokinin 6-benzylaminopurine (BAP) is able to increase PIN1 expression in pistils (Bencivenga et al., 2012). We therefore compared the number of ovules between wild-type mock-treated flowers at stage 8–9 and flowers treated with 1 mm BAP at 3 days after treatment. We observed an increase in ovule number in the BAP-treated pistils compared to the mock-treated ones (Figure 2g and Table S2). Similar treatments were performed using stage 8–9 pin1-5 flowers. In this case, we did not observe new ovule primordia in either the control or BAP-treated plants, confirming that PIN1 is required for the increase in ovule primordia in BAP-treated plants (Figure 2g and Table S2). These results are consistent with those reported by Bencivenga et al. (2012). Interestingly, we also observed an increase in ovule number in cuc2-1 pSTK::CUC1_RNAi carpels of BAP-treated inflorescences (Figure 2g and Table S2). The increase in ovule numbers in this genetic background (46.0%) was comparable with that obtained in treated wild-type plants (46.3%; Figure 2g and Table S2). To verify whether the phenotypic complementation observed in BAP-treated plants at 3 days after treatment was linked to the restoration of PIN1 expression levels, we analyzed pPIN1::PIN1-GFP levels (Figure S4) and performed quantitative RT-PCR (Figure 2e) in BAP- and mock-treated wild-type and cuc2-1 pSTK::CUC1_RNAi plants. This showed that expression of PIN1 is increased in BAP-treated plants (Figure 2e).
We also tested the effect of cytokinin on ovule numbers in the ant-4 mutant background. BAP treatment was unable to complement ovule number reduction (Figure 2g and Figure S4) even though PIN1 expression increased after the treatment (Figure 2e), suggesting that ANT may act in a CUC1/CUC2-independent pathway, as is also suggested by the genetic data.
The GFP signal driven by the DR5 promoter is not affected in cuc2-1 pSTK::CUC1_RNAi ovule primordia
To analyze whether the auxin response is compromised, we crossed cuc2-1 pSTK::CUC1_RNAi plants with the auxin-responsive reporter line pDR5rev::GFP (Benková et al., 2003). The GFP signal (reflecting auxin accumulation) was not visible in the placenta or in the ovule primordia at stage 1-I (Figure S5). However, the GFP signal was visible in the mutant ovule primordia starting from stage 1-II (Figure S5). This pattern of expression is identical to that of wild-type plants, suggesting that, although PIN1 expression is affected in these plants, the auxin maximum is nevertheless established in the ovule primordia.
It has been shown previously that DORNRÖSCHEN (DRN), which encodes an AP2 domain transcription factor, is transcriptionally regulated by auxin signaling. In the presence of auxin, use of the DRN regulatory region to drive GFP expression has been reported to be more responsive than use of the DR5 promoter (Chandler et al., 2011). Therefore, in an attempt to observe auxin-induced signals earlier during ovule development, we crossed cuc2-1 pSTK::CUC1_RNAi plants with pDRN::GFP plants. In both wild-type and cuc2-1 pSTK::CUC1_RNAi plants, the DRN promoter was active from stage 1-I, although we were unable to detect GFP before the ovule primordia were formed (Figure S5). Whilst these experiments do not allow us to verify whether PIN1 down-regulation in cuc2-1 pSTK::CUC1_RNAi plants affects the local auxin distribution in the placenta, we conclude that, once the primordia are formed, the auxin gradient along the ovule axis is not affected.
ANT, CUC1 and CUC2 are targets of MONOPTEROS
Analysis of the yuc1 yuc4 double mutant and the mp mutant suggests that auxin is required for ovule development (Cheng et al., 2006; Cole et al., 2009; Lohmann et al., 2010). The mp S319 mutant produces few flowers, all of which have a reduced number of organs, and, importantly, the pistils do not develop ovules (Cole et al., 2009; Lohmann et al., 2010). Morphological analysis shows that, in this mutant, the pistils do not have any carpel margin tissue (Figure S6). The placenta is completely missing and consequently ovules are unable to develop (Figure S6). To explore whether MP regulates CUC1, CUC2 and ANT expression in the pistil, we focused our analysis on its expression during the early stages of ovule development. We analyzed pMP::MP-GFP plants using confocal laser scanning microscopy. In these plants, GFP is visible in the placenta before ovule primordia are formed (Figure 3a). Once the primordia arise (stage 1-I), GFP expression is observed in the epidermal cell layer of the primordia (Figure 3b). Starting from stage 1-II, MP–GFP is mainly localized in the boundaries between the ovules (Figure 3c), and, from stage 2-I, is localized in the boundary between the nucellus and the chalaza (Figure S7).
Like MP, ANT (Figure 3d,e), CUC1 (Figure 3f,g) and CUC2 (Figure 3h,i) are expressed in the placenta before ovule primordia arise, and in ovules at stage 2-II. However, starting from stage 2-I, ANT is expressed in the chalaza and in the integument primordia, whereas CUC1 and CUC2 expression is limited to the boundary region between the chalaza and the nucellus, as is the case for MP (Figure S7). To study whether MP regulates ANT, CUC1 and CUC2 expression, we performed quantitative RT-PCR on pistils of the mp S319 mutant and the wild-type. This analysis revealed that ANT, CUC1 and CUC2 are down-regulated in this mutant (Figure 4a).
Interestingly, the rosette leaf margins of the mp S319 mutant show less serration than those of wild-type leaves (Figure 4b,c). This phenotype was previously described in the cuc2 mutant (Bilsborough et al., 2011). Consequently, we performed quantitative RT-PCR to analyze whether MP also controls CUC2 expression in leaves. As shown in Figure 4(d), CUC2 is down-regulated in mp S319 leaves, suggesting that MP also controls CUC2 expression in these organs.
It has recently been shown that ANT is a direct target of MP during floral meristem formation (Yamaguchi et al., 2013), and therefore it is likely that MP may also control ANT expression in the pistil. To assess whether MP directly regulates CUC1 and CUC2, we performed chromatin immuno-precipitation (ChIP) experiments using anti-GFP antibodies on chromatin extracted from the pistils of pMP::MP-GFP mp/mp plants before fertilization. The MP–GFP fusion protein fully complements the mp phenotype, indicating that the MP–GFP protein is biologically functional (Schlereth et al., 2010). Pre-fertilization wild-type pistils were used as a negative control, and ARABIDOPSIS RESPONSE REGULATOR15 (ARR15) and ANT, which are direct targets of MP, were used as positive controls (Zhao et al., 2010; Yamaguchi et al., 2013).
Within the genomic regions of CUC1 and CUC2, starting from 3 kb upstream of the ATG start codon to 0.5 kb downstream of the stop codon, several putative ARF binding sites were identified (Figure 5a). Three independent quantitative RT-PCR experiments were performed for each of the three independent immuno-precipitated chromatin samples. This analysis indicates that MP binds to the ARF binding site present in the CUC1 genomic region (Figure 5b), and also that present in CUC2 at 34 bp after the start codon (Figure 5a,b). No enrichment was evident in correspondence to the ARF binding site present at 1734 bp upstream of the CUC2 start site was evident (Figure 5b). These data support the contention that CUC1 and CUC2 may be direct targets of MP during pistil development.
Ovule primordia formation: an integrative model
Lateral organ formation requires the integrated action of hormones such as auxin and cytokinin, which have been shown to play a pivotal role in the control of organ development (Ruzicka et al., 2009). Although the local auxin response and auxin transport are required to form the placenta (Nemhauser et al., 2000), it appears that the cytokinin pathway-related genes play an important function in establishing ovule primordia formation (Werner et al., 2003; Kinoshita-Tsujimura and Kakimoto, 2011; Bencivenga et al., 2012). Once the primordium is formed, PIN1 is involved in re-establishing the auxin gradient along the axis of the newly developing organ, with a maximum occurring along the edge (Benková et al., 2003). We have previously shown that PIN1 expression and localization in ovules are controlled by cytokinin through the action of BELL1 (BEL1) and SPOROCYTELESS/NOZZLE (SPL/NZZ) (Bencivenga et al., 2012). However, these two transcription factors appear to be important for establishing the ovule pattern without influencing ovule number (Bencivenga et al., 2012), as they are expressed from stage1-I when the primordium is already formed (Schiefthaler et al., 1999; Balasubramanian and Schneitz, 2000). In roots, PIN gene expression and PIN protein localization are also controlled by cytokinin, which modulates cell-to-cell auxin transport and consequently auxin levels (Ruzicka et al., 2009).
That PIN1 is involved in the determination of ovule number was inferred from characterization of the pin1-5 mutant, as it develops fewer ovules compared to wild-type plants (Bencivenga et al., 2012). According to our data, CUC1 and CUC2 are required for both correct PIN1 expression and PIN1 localization. However, in cuc2-1 pSTK::CUC1_RNAi plants, we did not observe any changes in auxin accumulation during early stages of ovule development. It may be that DR5 is not sensitive enough to detect the changes. Involvement of CUC2 in PIN1 expression has already been proposed for the formation of leaf serrations (Bilsborough et al., 2011). Although we have shown that CUC1 and CUC2 are able to induce PIN1 expression in vivo using protoplast assays, it is likely that PIN1 transcriptional activation is indirect. Starting from stage 1-I, PIN–GFP is localized in cells (Ceccato et al., 2013;) in which CUC1 and CUC2 are not expressed, suggesting the involvement of non-cell-autonomous CUC downstream target(s) in the regulation of PIN1 expression.
The reduction in the number of ovules in cuc2-1 pSTK::CUC1_RNAi plants is less pronounced than that seen in the pin1-5 mutant, indicating that factors other than the CUC proteins regulate PIN1 expression at this stage of ovule development. Our study shows that treatment with BAP results in restored ovule numbers in cuc2-1 pSTK::CUC1_RNAi plants. However, BAP treatment does not have any effect if PIN1 is mutated, as is the case in pin1-5 plants, suggesting that cytokinin acts downstream of the CUC1 and CUC2 transcription factors to induce PIN expression. It will be of interest to determine whether this regulatory mechanism is also conserved in other organs such as leaves, in which it has been shown that a CUC2-dependent regulatory pathway controls PIN1-mediated auxin efflux (Bilsborough et al., 2011).
The results of BAP treatment suggested that PIN1 expression levels are important for ovule primordia formation, but it is not clear how PIN1 membrane localization is compatible with primordia formation and the observed maintenance of the auxin gradient during ovule development. The observation that BAP treatment cannot complement the ant-4 phenotype suggests that ANT functions in a pathway that is independent of CUC1 and CUC2. This is supported by the additive effects on the reduction in ovule numbers observed in ant-4 cuc2-1 pSTK::CUC1_RNAi plants.
MP is a key player in ovule primordia formation
MP is broadly expressed, and is involved in the transcriptional regulation of several auxin-responsive genes (Cole et al., 2009; Donner et al., 2009; Schlereth et al., 2010). Our data suggest that MP directly regulates the expression of ANT, CUC1 and CUC2 during the formation of placenta and ovule primordia. Accordingly, expression of these genes in the pistil is reduced in a partial loss-of-function mp mutant (mp S319). The role of ANT in pistil development has been described previously. For example, in ant lug and ant seuss double mutants, as well as in the ant shp1 shp2 crc quadruple mutant, placenta formation is seriously compromised and ovules are not formed (Liu et al., 2000; Azhakanandam et al., 2008; Colombo et al., 2010; Wynn et al., 2011). We suggest that, in the pistil, MP responds to auxin by activating the expression of ANT, which is required for correct pistil and ovule primordia formation. In this regard, it has recently been suggested that MP also regulates ANT expression in the floral meristem (Yamaguchi et al., 2013). Furthermore, we suggest that MP may regulate the expression of CUC1 and CUC2 in the pistil, which is required for PIN1 expression.
Due to the lack of placenta tissue in the mpS319 mutant, it is not possible to discriminate between the effect of MP on ANT and CUC proteins in the placenta with respect to ovule primordia formation, although it is clear that MP is required for ANT, CUC1 and CUC2 expression in the pistil, as shown by RT-PCR experiments. The ChIP experiments suggest that MP directly regulates the transcription of ANT, CUC1 and CUC2, although functional analysis of the binding site is required to verify this suggestion.
Based on our results, we propose a model in which MP regulates ANT, CUC1 and CUC2 expression during the early stages of placenta and ovule development (Figure 6). The expression of CUC1 and CUC2 is necessary for correct PIN1 expression, which, in turn, is a prerequisite for ovule primordia formation. Once PIN1 is expressed, an auxin responce occurs in the apex of the nucellus (Figure 6). The high auxin concentration may repress CUC genes, as shown previously for CUC2 in leaves (Bilsborough et al., 2011). This model is very similar to that proposed for the development of leaf serration (Bilsborough et al., 2011). At later stages of development, expression of MP and the CUC genes is confined to the boundary regions between ovules and the region between the nucellus and the chalaza. Once ovules have formed, the interaction between auxin and cytokinin provides a signaling system for the correct growth and development of ovules (Bencivenga et al., 2012; Marsch-Martínez et al., 2012). Interestingly, our results appear to confirm the importance of cytokinin in controlling ovule numbers (Werner et al., 2003; Kinoshita-Tsujimura and Kakimoto, 2011; Bencivenga et al., 2012), and suggest that interaction between the auxin and cytokinin pathways is required for formation of ovule primordia.
Arabidopsis thaliana wild-type and mutant plants were grown at 22°C under short-day conditions (8 h light per 16 h dark) or long-day conditions (16 h light per 8 h dark) conditions. ant-4 (Baker et al.,1997), cuc1-1 (Takada et al., 2001) and cuc2-1 (Aida et al., 1997) mutant seeds were obtained from the Nottingham Arabidopsis Stock Center (arabidopsis.info). pDRN::GFP (Cole et al., 2009) was obtained from Wolfgang Werr (Institut für Entwicklungsbiologie, University of Cologne, Germany). pMP::SV40-3xGFP (Rademacher et al., 2011), pMP::MP:GFP mp-5/mp-5 (Schlereth et al., 2010) and mp S319 (Cole et al., 2009) have been described previously.
BAP treatment was performed on flowers at stage 8–9 of development, as previously described by Bencivenga et al. (2012). We collected treated inflorescences at 3 days after treatment, and counted the numbers of ovules in pistils.
Optical and confocal microscopy
GUS staining was performed overnight as described previously (Liljegren et al., 2000). Siliques and carpels were collected and cleared as described by Yadegari et al. (1994). Pistils were observed using a Zeiss Axiophot D1 microscope (http://www.zeiss.com) equipped with differential interference contrast optics. Images were recorded using an Axiocam MRc5 camera (Zeiss) with Axiovision version 4.1.
For confocal laser scanning microscopy, fresh material was collected, mounted in water and immediately analyzed. Confocal laser scanning microscopy analysis was performed using a Leica (www.leica-microsystems.com) TCS SPE microscope with a 488 nm argon laser line for excitation of GFP fluorescence. Emissions were detected between 505 and 580 nm. Images were collected in multi-channel mode, and overlay images were generated using Leica analysis software LAS AF 2.2.0.
Plasmid construction and Arabidopsis transformation
To construct pSTK::CUC1_RNAi, a specific CUC1 fragment (nucleotides 730–925) was amplified using primers AtP_2916 and AtP_2917, and recombined into RNAi vector pFGC5941 (Karimi et al., 2002) through an LR reaction (Gateway system, Invitrogen, www.lifetechnologies.com). The CaMV 35S promoter of the pFGC5941 vector was removed and substituted by the STK promoter (amplified using primers AtP_590 and AtP_591) (Kooiker et al., 2005). This construct was used to transform ant-4 cuc2-1 plants using the floral-dip method (Clough and Bent, 1998). The primers used are listed in Table S3.
RT-PCR and quantitative RT-PCR analysis
Total RNA was extracted from pistils at stages 8–10 of flower development (Roeder and Yanofsky, 2006) using the LiCl method (Verwoerd et al., 1989). Total RNA was treated using the Ambion TURBO DNA-free DNase (http://www.lifetechnologies.com/it/en/home/brands/ambion, kit and then reverse transcribed using the ImProm-II™ reverse transcription system (Promega, www.promega.com). The cDNAs were standardized relative to UBIQUITIN10 (UBI10) and ACTIN2-8 (ACT2-8) transcripts, and gene expression analysis was performed using the iQ5 Multicolor real-time PCR detection system (Bio-Rad, www.bio-rad.com) with SYBR Green PCR Master Mix (Bio-Rad). Baseline and threshold levels were set according to the manufacturer's instructions. RT-PCR and quantitative RT-PCR primers are listed in Table S1.
Protoplast preparation and transient expression experiments were performed as described by De Sutter et al. (2005). Protoplasts were prepared from a BY-2 tobacco cell culture, and co-transfected using a reporter plasmid containing the Firefly LUCIFERASE (fLUC) reporter gene driven by the PIN1 or PIN7 promoter, together with a normalization construct expressing Renilla LUCIFERASE (rLUC) and effector constructs expressing CUC1 and CUC2, respectively, under the control of the CaMV 35S promoter. The reporter construct was generated as follows: the pEN-L4-PIN1-R1 or pEN-L4-PIN7-R1 vector (where the PIN1 promoter comprises −2098 bp upstream of the PIN1 coding sequence, and the PIN7 promoter comprises −1423 bp upstream of the PIN7 coding sequence) was recombined together with pEN-L1-fLUC-L2 by multi-site Gateway LR cloning with pm42GW7 (Karimi et al., 2007). For the effector constructs, pEN-L1-‘ORF’-R2 [where ‘ORF’ represents CUC1 genomic DNA (1565 bp) or the CUC2 coding sequence (1128 bp)] was used to introduce the ORFs into p2GW7 (Karimi et al., 2002) by Gateway LR cloning for over-expression. Two micrograms of each construct were added, and the total effector amount was equalized in each experiment using the p2GW7-GUS mock effector plasmid. After transfection, protoplasts were incubated overnight and then lysed; fLUC and rLUC activities were determined using the dual-luciferase reporter assay system (Promega). Variations in transfection efficiency and technical error were corrected by normalization of fLUC against rLUC activities. All transactivation assays were performed in an automated experimental set-up that involved eight separate transfection experiments, and were performed at least twice.
Chromatin immunoprecipitation (ChIP) assays
ChIP experiments were performed using a modified version of a previously described protocol (Gregis et al., 2008) using the commercial antibody GFP:Living Colors full-length A.v. polyclonal antibody (Clontech, www.clontech.com). Chromatin was extracted from stage 8–10 pistils of pMP::MP-GFP plants and from wild-type plants (Col-0) as a control. The DNA fragments obtained from the immune-precipitated chromatin were amplified by quantitative RT-PCR using specific primers (see Table S1). Three real-time PCR amplifications on three independent chromatin extractions were performed. The primers are listed in Table S1. Enrichment of the target region was determined using the iQ5 Multicolor real-time PCR detection system (Bio-Rad) with SYBR Green PCR Master Mix (Bio-Rad). The quantitative RT-PCR assays and the fold enrichment calculation were performed as described previously (Matias-Hernandez et al., 2010).
In situ hybridization
In situ hybridization experiments were performed as previously described (Dreni et al., 2011). The ANT, CUC1 and CUC2 probes were amplified as described by Elliott et al. (1996) for ANT and Aida et al. (2002) for CUC1 and CUC2, and subsequently cloned into the pGEMT-Easy vector (Promega) (Table S1).
The project and F.G. were supported by the CARIPLO Foundation (project 2009-2990) and COST (European Cooperation in Science and Technology) action HAPRECI (Harnessing Plant Reproduction for Crop Improvement). E.B. and C.C. were supported by the European Research Council through a ‘Starting Independent Research’ grant (ERC-2007-Stg-207362-HCPO). We thank A.P. MacCabe (Consejo Superior de Investigaciones Científicas, Valencia, Spain) for critical reading of the manuscript.