SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein
1999; Springer Nature; Volume: 18; Issue: 15 Linguagem: Inglês
10.1093/emboj/18.15.4169
ISSN1460-2075
Autores Tópico(s)Plant Virus Research Studies
ResumoArticle2 August 1999free access SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein Catherine Delichère Catherine Delichère Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France Present address: Laboratoire de Physiologie Cellulaire Végétale, DBMS-CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, cedex 9, France Search for more papers by this author Jacky Veuskens Jacky Veuskens Institute of Molecular Cell Biology, University of Amsterdam, 1098 SM, Amsterdam, The Netherlands Search for more papers by this author Michel Hernould Michel Hernould Université Bordeaux II, Laboratoire de Biologie Cellulaire, Avenue des Facultés, 33405 Talence, France Search for more papers by this author Nicolas Barbacar Nicolas Barbacar Institute of Genetics, Academy of Sciences, 277018 Chisinau, Republic of Moldavia Search for more papers by this author Armand Mouras Armand Mouras Université Bordeaux II, Laboratoire de Biologie Cellulaire, Avenue des Facultés, 33405 Talence, France Search for more papers by this author Ioan Negrutiu Corresponding Author Ioan Negrutiu Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France Search for more papers by this author Françoise Monéger Françoise Monéger Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France Search for more papers by this author Catherine Delichère Catherine Delichère Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France Present address: Laboratoire de Physiologie Cellulaire Végétale, DBMS-CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, cedex 9, France Search for more papers by this author Jacky Veuskens Jacky Veuskens Institute of Molecular Cell Biology, University of Amsterdam, 1098 SM, Amsterdam, The Netherlands Search for more papers by this author Michel Hernould Michel Hernould Université Bordeaux II, Laboratoire de Biologie Cellulaire, Avenue des Facultés, 33405 Talence, France Search for more papers by this author Nicolas Barbacar Nicolas Barbacar Institute of Genetics, Academy of Sciences, 277018 Chisinau, Republic of Moldavia Search for more papers by this author Armand Mouras Armand Mouras Université Bordeaux II, Laboratoire de Biologie Cellulaire, Avenue des Facultés, 33405 Talence, France Search for more papers by this author Ioan Negrutiu Corresponding Author Ioan Negrutiu Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France Search for more papers by this author Françoise Monéger Françoise Monéger Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France Search for more papers by this author Author Information Catherine Delichère1,2, Jacky Veuskens3, Michel Hernould4, Nicolas Barbacar5, Armand Mouras4, Ioan Negrutiu 1 and Françoise Monéger1 1Ecole Normale Supérieure de Lyon, Laboratoire de Reproduction et Développement des Plantes, UMR 5667 CNRS/INRA/ENS/Lyon I, 46 Allée d'Italie, 69364 Lyon, France 2Present address: Laboratoire de Physiologie Cellulaire Végétale, DBMS-CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, cedex 9, France 3Institute of Molecular Cell Biology, University of Amsterdam, 1098 SM, Amsterdam, The Netherlands 4Université Bordeaux II, Laboratoire de Biologie Cellulaire, Avenue des Facultés, 33405 Talence, France 5Institute of Genetics, Academy of Sciences, 277018 Chisinau, Republic of Moldavia *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:4169-4179https://doi.org/10.1093/emboj/18.15.4169 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y-specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y-gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants. Introduction Although the majority of plants differ from animals in that they are hermaphrodite, a small proportion have separate sexes; they are dioecious (Renner and Ricklefs, 1995). In some of these dioecious species, sex determination is controlled by heteromorphic sex chromosomes. Sex determination systems based both on the X/autosome ratio (as in Drosophila) and on X/Y with a dominant Y chromosome (as in mammals) exist in the plant kingdom (Parker, 1990; Ainsworth et al., 1998). This convergent evolution of sex determination mechanisms raises exciting and challenging questions about the structure, function and evolution of sex chromosomes in the two kingdoms. While genetic and molecular mechanisms controlling sex determination in Drosophila and mammals have been studied extensively (Nöthiger and Steinmann-Zwicky, 1987; Laurie, 1997; Capel, 1998; Raymond et al., 1998), very little is known about such processes in plants. White campion (Silene latifolia) is one of the most suitable plants to address such questions since in this species, sex determination is, as in mammals, under the strict genetic control of a dominant Y chromosome (Westergaard, 1958; Van Nigtevecht, 1966). XX individuals develop female flowers, whereas XY individuals develop male flowers. The presence of a single Y chromosome is sufficient to induce the development of male flowers, even in the presence of several X chromosomes (Westergaard, 1958), demonstrating the active nature of this chromosome. Genetic and cytogenetic studies have shown that the Y chromosome plays a key role in sex determination in this species. At a very early stage, floral meristems from both sexes are potentially hermaphrodite: they contain both male and female reproductive organ primordia, as shown in Figure 1. Sex determination is under the control of two independent functions located on the Y chromosome: the first blocks gynoecium (female organ) development and the second promotes stamen (male organ) development. As a result, in a male XY plant, flowers develop with stamens and an undifferentiated filamentous structure instead of the gynoecium in the center of the flower (Figure 1). Conversely, in a female XX plant, flowers develop with a normal gynoecium but stamens stop developing at an early stage (Farbos et al., 1997). The Y chromosome also carries male fertility genes (Figure 1), since male sterile mutants with deleted Y chromosomes have been reported (Westergaard, 1958; Donnison et al., 1996). Finally, the X and Y chromosomes share a pseudo-autosomal region (PAR) through which the two chromosomes specifically pair during meiosis (Westergaard, 1958; Buzek et al., 1997). Our knowledge of the Y chromosome is limited to morphological, genetic and cytogenetic data. Apart from genes involved in sex determination and male fertility, are there other active genes on this chromosome? Does it contain housekeeping genes as has been shown to be the case for the human Y chromosome (Lahn and Page, 1997)? What is the extent of homology between the S.latifolia X and Y chromosomes ? Figure 1.Sex determination and Y chromosome in S.latifolia. At a very early stage of development, the floral meristem is similar to that of a hermaphrodite species and contains four types of organ primordia: sepal (1), petal (2), male organ (3) and female organ (4). The subsequent developmental fate of the floral meristem depends on the presence or absence of the Y chromosome. In an XY background, female organ (gynoecium, gy) development is blocked by the female suppression function on the Y chromosome (♀−) giving rise to a filament (f), and the male organ (stamens, st) development is activated by the male promotion function on the Y chromosome (♂+). Finally, the male fertility genes located on the Y chromosome are involved in pollen development. In an XX background, the gynoecium develops normally, as a default state, and stamens stop developing at a very early stage due to lack of activation. PAR, pseudo-autosomal region. Download figure Download PowerPoint Several attempts to identify active genes located on the Y chromosome have resulted in the isolation of non-coding sequences, the vast majority of which are shared by Y, X and/or autosomes (Donnison et al., 1996; Scutt et al., 1997; Zhang et al., 1998). In this paper, we report the characterization of the first active gene cloned from a plant Y chromosome. We microdissected Y chromosomes from S.latifolia and a Y chromosome-specific probe was used to identify five cDNA clones corresponding to Y-linked genes. Here, we describe the detailed analysis of one of these clones, SlY1 (S.latifolia Y-gene 1). We show that it is linked to the Y chromosome and that a closely related gene is most likely located on the X chromosome (SlX1). SlY1 and SlX1 are highly homologous and both genes are expressed. They encode WD-repeat proteins that are localized in the nucleus and which are most abundant in cells that are actively dividing or beginning to differentiate. Taken together, these results allow us to draw preliminary parallels between the human and a plant Y chromosome. Finally, we envisage the use of SlY1 and SlX1 in the cloning of the sex determination genes in white campion. Results Screening of an early male flower cDNA library with a Y-derived probe In order to characterize expressed genes located on the Y chromosome, Y chromosomes were microdissected on metaphase spreads from root cultures. Two pools of 10 microdissected Y chromosomes were used as template for Degenerate Oligonucleotide Primed–PCR amplification (DOP–PCR). Each pool was amplified with one of two primers (Materials and methods) in two successive PCR amplifications. The sizes of the amplicons ranged from ∼0.2 to 4 kb (data not shown). The PCR products were pooled to produce the Y-derived probe (Materials and methods) and used to screen a premeiotic male flower cDNA library (Barbacar et al., 1997). We selected 115 positive clones showing different signal intensities. In order to test for Y-linkage, segregation analysis was performed: each insert was hybridized with restricted genomic DNA from male and female individuals as well as their male and female progeny. Three kinds of profiles were obtained: (i) clones showing no difference between males and females; (ii) clones showing restriction fragment length polymorphism; and (iii) clones showing sex-linked polymorphism. In total, we identified five partial cDNA clones hybridizing to one or more male-specific fragment(s). In this paper, we report the detailed analysis of one of these cDNAs named SlY1. A genomic DNA fragment located on the Y chromosome contains the gene encoding the SlY1 cDNA In order to confirm the Y-linkage of SlY1, segregation analysis was performed by genomic Southern blot analysis. HindIII-restricted genomic DNA from two parents (F0) and 28 F1 progeny plants (14 males and 14 females) were tested. The results obtained for 10 representative plants are shown in Figure 2. A portion of the SlY1 cDNA (probe B, Figure 3A) recognized two bands of 12 and 6 kb, respectively, which were present in both male and female DNA, but also a band of 4.5 kb which was only detected in DNA from the male plants (Figure 2). The probability that this strict segregation could be a random event was extremely low: one chance in 3.7×109 [P = (1/2)n, where n is the number of segregating male and female individuals for the fragment of interest]. These results demonstrate that the 4.5 kb HindIII fragment is linked to the Y chromosome. Figure 2.Segregation analysis of a male-specific fragment and characterization of SlY1. Genomic DNA isolated from two parent plants (F0 male and F0 female) and from their male (F1 male 1–4) and female (F1 female 1–4) progeny was digested with HindIII and analysed by Southern blot. Genomic DNA was hybridized with a restriction fragment derived from the SlY1 cDNA (probe B, Figure 3A). The size of the fragments which hybridize to the probe are indicated. The 4.5 kb band which segregates with the male sex is labelled with a Y (left). Download figure Download PowerPoint Figure 3.Characterization of gene-specific probes for SlY1 and SlX1 respectively. (A) The cDNA sequences of SlY1 and SlX1 are represented with their coding regions boxed in grey and black respectively. The primers used for PCR are shown by arrowheads. The percentage of homology between the coding regions and part of the 3′ untranslated regions of the two genes are indicated. The four fragments (A, B, C, D) used as probes in the Southern blot analysis in panel (B) are represented by dark lines. Probe A is a 1192 bp RT–PCR product amplified from SlY1 cDNA using primers S1 and AS9, probe B corresponds to a 273 bp Sau3AI subfragment from the SlY1 cDNA, probe C is a 107 bp RT–PCR product amplified from SlY1 cDNA using the primers S11 and AS9 primers, probe D is a 117 bp RT–PCR product amplified from SlX1 cDNA using the primers S12 and AS10 primers. The scale is indicated by a bar (bottom left) representing 100 bp. SlY1 and SlX1 sequences are available in the DDBJ/EMBL/GenBank databases under accession Nos Y18517 and Y18519 respectively. (B) Genomic DNA isolated from a male (♂) or a female (♀) plant was digested with HindIII and analyzed by Southern blotting. The same DNA samples were hybridized with four different probes [A–D, panel (A)] as indicated at the top of each blot. The fragments labelled with a triangle correspond to the SlY1 locus. The fragments labelled with a circle correspond to the SlX1 locus. The size of some fragments are indicated. (C) Genomic DNA from male or female individuals was used as template in PCR amplification using either the S11 and AS9 primers specific for SlY1, or the S12 and AS10 primers specific for SlX1 (panel A). The PCR products were loaded on an agarose gel and separated by electrophoresis in the presence of ethidium bromide. DNA fragments were visualized under UV light. (D) Genomic DNA isolated from male (♂) or female (♀) plants, or from four Y chromosome deletion mutants exhibiting hermaphrodite (bsx) or asexual (asx) phenotypes, was digested with HindIII and analyzed by Southern blot using probe B (panel A). The origin of the fragments are indicated on the right. SlX1 exhibits an allelic polymorphism in the asexual mutants. On the left, the Y chromosome is represented with its p and q arms. The black box represents the pseudo-autosomal region. Vertical bars represent the regions covered by the deletions in each type of mutant. Download figure Download PowerPoint Identification of two highly homologous cDNAs, SlY1 and SlX1 The initial SlY1 cDNA was 706 bp long and was truncated at both the 5′ and 3′ ends. In order to obtain a complete SlY1 cDNA, we performed rapid amplification of cDNA ends (RACE–PCR). The primers used for the RACE–PCR experiments are shown in Figure 3A. The sense primer S7 was used to amplify two distinct 3′ cDNA sequences. The 5′ region of the SlY1 cDNA, including the putative initiation codon of a 1416 bp open reading frame, was amplified with the antisense primers AS6 and AS13. We used RT–PCR amplifications with primers S14 and AS9 to verify the continuity of the SlY1 cDNA clone. Similarly, RT–PCR with primers S14 and AS10 confirmed the existence of a related cDNA named SlX1. The sequences of each of these two cDNAs were determined from PCR products obtained from at least two independent RT–PCR experiments. These two cDNAs are represented in Figure 3A were respectively 1785 (SlY1) and 1739 (SlX1) bp long (not including their polyA tails). The two putative coding sequences are 99% homologous at the DNA level. In the 3′ untranslated region, the two cDNAs are 90.8% homologous, with the exception of a region of 100 bp at the extreme 3′ end where they diverge completely. In order to determine the chromosomal origin of each of these two cDNAs, Southern blot analysis was performed using different DNA fragments as probes against HindIII-restricted genomic DNA. The probes are shown in Figure 3A, and corresponding Southern blots are shown in Figure 3B. Probe B hybridized to a male-specific, 4.5 kb fragment and, more weakly to 12- and 6-kb fragments present in DNA from both males and females. Hybridization to the 12 kb fragment was twice as strong in DNA from females compared with DNA from males (probes A and B) as would be expected for a X-linked gene. Probe A includes the probe B region and extends into the 3′ untranslated region of the SlY1 cDNA. It hybridized to more fragments than probe B. One of the male-specific fragments was 11 kb long and also hybridized to the SlY1-specific probe C (Figure 3A and B). The male-specific fragments corresponding to SlY1 (4.5 and 11 kb) are indicated with an arrowhead. The SlX1-specific probe D hybridized twice as strongly to a 1.2 kb fragment in DNA from females compared with DNA from males (Figure 3A and B). This supports the hypothesis that SlX1 is linked to the X chromosome. Therefore, we named this gene SlX1 (for S.latifolia X-gene 1). The fragments corresponding to SlX1 (12 and 1.2 kb) are indicated with a circle. Finally, we noted a complex hybridization pattern with probe A, indicating the existence of additional homologous genes within the genome. Nine restriction fragments were revealed by probe A in DNA from both males and females (Figure 3B panel A). Among these, two correspond to SlX1 and are labelled with a circle. Three others showed a double intensity in the DNA from females suggesting they could correspond either to SlX1 (since the locus has not yet been sequenced completely) or to other homologues located on the X chromosome. The other four (one of them had the same size as the 4.5 kb fragment corresponding to SlY1) probably corresponded to homologues located on autosomes. Five fragments were detected only in DNA from the males. Based on the gene sequence (data not shown), two of them were assigned to SlY1 and are indicated with an arrowhead. The other three could correspond to homologue(s) also located on the Y chromosome. Their analysis is underway. The specificity of the primers, S11 and AS9 for SlY1, and S12 and AS10 for SlX1 (Figure 3A), was confirmed by PCR amplification of genomic DNA from male and female individuals. The results are shown in Figure 3C: primers S11 and AS9 amplified a 107 bp fragment only when DNA from a male plant was used as a template, whereas S12 and AS10 amplified a 117 bp fragment with DNA from both males and females. The intensity of the band with the DNA from the female was stronger than with the DNA from the male. This is in agreement with X-linkage of SlX1. In order to establish whether SlY1 maps to regions of the Y chromosome known to contain sex determination loci, we performed genomic Southern blot analysis on sexual mutants obtained by γ-irradiation of pollen grains. These mutants have been shown to have deleted Y chromosomes (Farbos et al., 1999; Lardon et al., 1999). The hermaphrodite mutants have lost the female suppressing function, whereas the asexual mutants have lost the male promoting function. Both of these functions are located on the Y chromosome and are responsible for sex determination (Figure 1). We chose to test four mutants with the largest reported Y chromosome deletions: two hermaphrodite mutants bsx1 (51% of the differential arm deleted) and bsx2 (38% of the differential arm deleted) (Lardon et al., 1999), and two asexual mutants asx1 (40% of the differential arm deleted) and asx2 (24% of the differential arm deleted) (Farbos et al., 1999). Total HindIII-restricted genomic DNA from male, female and mutant plants was hybridized with probe B (Figure 3A and B) and the results are shown in Figure 3D. As expected, the 4.5 kb fragment corresponding to the SlY1 gene was present in the DNA from the male plant but not from the female plant. All the mutants tested contained this 4.5 kb fragment, indicating that SlY1 is not deleted in any of them. We concluded that SlY1 does not map to the regions covered by these deletions. Note that SlX1 seemed to exhibit allelic polymorphism in the two asexual mutants, probably due to the different origin of the female parent compared with hermaphrodite mutants. All Y chromosome deletion mutants received the X chromosome from the non-irradiated mother. SlY1 and SlX1 encode WD-repeat proteins Both SlY1 and SlX1 cDNAs contained open reading frames encoding putative polypeptides of 472 amino acids (52 kDa). The sequences of these two polypeptides are 99.6% identical and differ at only two amino acid positions: position 154, residue Trp for SlY1 and Leu for SlX1, and position 441, Gly for SlY1 and Arg for SlX1. The sequences were established from independent PCR products in order to rule out PCR errors. These differences may or may not have consequences on the specificity of interaction of each protein with potential partners. When the SlY1 protein sequence was compared with the database, homology with members of the WD-repeat protein family was revealed. A typical core element of a WD-repeat is (G,V,A,N,S,I)H-X28-(W,F)(D,S), where X stands for any amino acid (Neer et al., 1994). Such proteins are found almost exclusively in eukaryotes, where they are involved in a variety of processes including regulation of transcription, control of cell growth and differentiation, and chromatin structure (Neer et al., 1994; Neer and Smith, 1996; Mulligan and Jacks, 1998; Parkhurst, 1998). Significant similarities ranging from 30 to 42% were found with proteins from different organisms: RbAp46 and RbAp48 from human (Qian et al., 1993; Qian and Lee, 1995); MSI1 and Hat2p from yeast (Ruggieri et al., 1989; Parthun et al., 1996); the p55 subunit of dCAF-1 from Drosophila (Kamakaka et al., 1996); and MSI1-like proteins recently reported in plants: LeMSI1 from tomato and AtMSI1, AtMSI2 and AtMSI3 from Arabidopsis thaliana (Ach et al., 1997). Another MSI1-like protein from A.thaliana, named AtMSI4 (Kenzior and Folk, 1998), is very homologous to the SlY1 protein, sharing 82% similarity (77% identity) with it. In Figure 4A, we show an amino acid alignment between the SlY1 and AtMSI4 proteins. The five highly conserved WD-repeats are boxed as well as the two amino acids flanking the core element. Figure 4.SlY1 encodes a WD-repeat protein. (A) Alignment of the protein sequences of SlY1 with that of AtMSI4 from A.thaliana. The two proteins are 77% identical. Vertical bars represent identical amino acids, single and double dots represent similar amino acids. The five WD-repeat domains are boxed, as well as the two amino acids flanking this core: GH, IH or AH at the beginning, and WD, WS or FD at the end (Neer et al., 1994). (B) Phylogenetic tree based on an alignment of SlY1 and 10 related genes. The 10 SlY1-related proteins are members of the WD-repeat family and include proteins from tomato (LeMSI) (Ach et al., 1997), A.thaliana (AtMSI1, AtMSI2, AtMSI3 (Ach et al., 1997) and AtMSI4 (Kenzior and Folk, 1998), human RbAp48, RbAp46 (Qian et al., 1993; Qian and Lee, 1995), Drosophila p55 subunit of dCAF-1 (Kamakaka et al., 1996) and yeast MSI1 (Ruggieri et al., 1989), Hat2 (Parthun et al., 1996). Protein sequences were aligned using the MUST package (Philippe, 1993). Distance trees were calculated using the neighbour-joining method. The length of the branches are proportional to the degree of divergence and thus correspond to the statistical significance of the phylogeny between the protein sequences. The bootstrap values supporting the branches are indicated and were calculated using 1000 replicates. Download figure Download PowerPoint We selected 11 members of the WD-repeat family from plants, human, Drosophila and yeast with which SlY1 shares the highest levels of homology. The deduced protein sequences were aligned and Figure 4B shows the corresponding phylogenetic tree. Among these proteins, SlY1 and AtMSI4 define a separate group. The phylogenetic tree indicates a common origin for SlY1 and AtMSI4. Interestingly, proteins that have been shown to bind the Rb (Retinoblastoma protein), namely RbAp48, RbAp46, LeMSI1, and their close homologues, AtMSI1 and dCAF-1, form a phylogenetic branch distinct from the other members of the family. The alignment of these 11 proteins not only identified strong homologies in the first three WD-repeat domains (for example 65% homology between SlY1 and RbAp48), but also in the N- and C-terminal regions (yeast genes excluded), suggesting that in addition to the WD-repeats, the conserved N- and C-terminal regions may be important for the activity of these proteins in higher eukaryotes. Transcripts of both SlY1 and SlX1 accumulate in young flowers In order to investigate the expression profile of each of these two genes, the gene-specific probes C and D (Figure 3A and B) were used to perform Northern blot analysis on total RNA from different tissues. The developmental stages of the flowers are described in Materials and methods. The results are shown in Figure 5A. The SlY1 transcript (∼2.2 kb) was detected in male flowers at all stages of development tested but not in the vegetative tissues tested (leaves, stems and seedlings). Stamens harvested at stage 3 also contain relatively abundant SlY1 transcripts. As expected, no transcript was detected in tissues from females since the gene is absent. The SlX1 transcript (∼2.2 kb) was detected in male and female flowers at all stages of development tested, including gynoecium tissues at pre-meiotic stages where it was most abundant. A small amount of SlX1 transcript was detected in leaves and stems of female plants as well as in seedlings. As a control, a 1.9 kb transcript was detected by a fragment corresponding to the actin gene from white campion, confirming the presence of hybridizable RNA in all samples. These results were confirmed by RT–PCR amplification which allows a more sensitive detection, and are shown in Figure 5B. The primers used for the RT–PCR amplifications are shown in Figure 3A. The sizes of the amplification products were as expected: 1.2 kb for primers S1 and AS9 (SlY1), and 1.21 kb for primers S1 and AS10 (SlX1). The results were in agreement with the Northern blot analysis: SlY1 was only expressed in tissues from male plants and the transcript was mainly detected in male flowers and in stamens. A very small amount of SlY1 transcript was detected in leaves and stems, whereas it was not detected in seedlings (a mixture of male and female seedlings). SlX1 was expressed in both male and female tissues, with preferential expression in flowers of both sexes. However, the transcript was more abundant in female flowers. A smaller amount was detected in non-floral tissues of both sexes (leaves, stems, seedlings). As a control, two primers specific for actin genes were used and amplified a 0.7 kb fragment from all the samples. Figure 5.Expression of SlY1 and SlX1. (A) Northern blot analysis. Flower developmental stages are as described in Materials and methods. Fifteen microgrammes of total RNA from different tissues of male (♂) or female (♀) plants were loaded in each lane: flowers at three successive stages of development (Fb1, Fb2 and Fb3), leaves (L), stems (S), isolated male organs at stage 3 (St3), isolated female organs at stage 3 (Gy3) and seedlings (Se). The RNAs were hybridized with the three different probes indicated on the right. SlY1 corresponds to the SlY1-specific fragment (probe C, Figure 3A). SlX1 corresponds to the SlX1-specific fragment (probe D, Figure 3A). Actin corresponds to a fragment of actin cDNA amplified from S.latifolia with the actin-specific primers (Materials and methods). On the left, the sizes of the transcripts are indicated in kb. (B) RT–PCR analysis. Total RNA from the same tissues as in (A) were used as template for reverse transcription from an oligo-dT primer and subsequent PCR amplification using three different pairs of primers: S1 and AS9 to detect SlY1 transcripts, S1 and AS10 to detect SlX1 transcripts (Figure 3A) and actin-specific primers as a control (Materials and methods). The PCR products were separated on an agarose gel and stained with ethidium bromide. Their sizes are indicated on the left in kb. (C) Western blot analysis. Purified anti-SlY1/SlX1 antibody was used for immunodetection on 10 μg of total prote
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