| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
First published online July 15, 2005; 10.1104/pp.105.062059 Plant Physiology 138:2185-2199 (2005) © 2005 American Society of Plant Biologists SERK and APOSTART. Candidate Genes for Apomixis in Poa pratensis1,[w]Department of Plant Biology and Agro-Environmental and Animal Biotechnology, University of Perugia, 06121 Perugia, Italy (E.A., G.M., L.R., F.F., M.F.); Department of Environmental Agronomy and Crop Science, University of Padova, Agripolis, 35020 Legnaro, Padova, Italy (G.B.); and Institute of Plant Genetics, Research Division of Perugia, 06100 Perugia, Italy (A.P.)
Seed production generally requires the mating of opposite sex gametes. Apomixis, an asexual mode of reproduction, avoids both meiotic reduction and egg fertilization. The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A more profound knowledge of the mechanisms that regulate reproductive events would contribute fundamentally to understanding the genetic control of the apomictic pathway. In Poa pratensis, we isolated and characterized two genes, PpSERK (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE) and APOSTART. These full-length genes were recovered by rapid amplification of cDNA ends and their temporal and spatial expression patterns were assessed by reverse transcription-polymerase chain reaction and in situ hybridization, respectively. The expression of PpSERK and APOSTART differed in apomictic and sexual genotypes. Their putative role in cell-signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis, and embryogenesis in plants is reported and discussed. We propose that, in nucellar cells of apomictic genotypes, PpSERK is the switch that channels embryo sac development and that it may also redirect signaling gene products to compartments other than their typical ones. The involvement of APOSTART in meiosis and programmed cell death is also discussed.
Sex is the queen of problems in evolutionary biology. Perhaps no other natural phenomenon has aroused so much interest; certainly none has caused as much confusion (Bell, 1982  ). One of the major questions is as follows: What is the meaning and the rationale of sexuality, which leads to its maintenance under natural selection in biological populations? The easiest answer is that sex, through genetic recombination at meiosis and genomic fusion by fertilization, allows genotype rearrangement, diversification, and adaptation. But even the most dogmatic interpretation of the meaning of sex can be challenged. For example, the existence of apomixis, which is reproduction through seeds but without sex, can be an argument that questions the necessity of sex. In apomictic plants, the embryo is fertilization independent and develops inside an embryo sac formed from either a megaspore mother cell (MMC; diplospory) or a somatic cell of the nucellus (apospory) and retains the maternal genotype (Savidan, 2000; Grimanelli et al., 2001; Spillane et al., 2001). Fertilization may be required for endosperm formation, which can either retain the 2:1 maternal:paternal genome ratio or completely lack a paternal genome equivalent (Nogler, 1984; Spillane et al., 2001; Tucker et al., 2003).
Defining the nature and genetic control of apomixis may be crucial for both understanding the trait itself and better illustrating the meaning of sexuality. Although many years of descriptive studies have provided a solid documentation of the types of apomictic processes that occur in a wide variety of plant species, molecular studies aimed at understanding the basis of apomixis have failed to shed more than a dim and wavering light on its central mystery, partly because the majority of apomicts do not constitute agriculturally important crops and, with few exceptions (e.g. Tripsacum and maize [Zea mays]), do not have agriculturally important relatives (Bicknell and Koltunow, 2004
Chaudhury and Peacock (1993)
Comparative gene expression studies have been carried out during the early stages of apomictic and sexual embryo sac development in Panicum maximum (Chen et al., 1999
Since natural apomixis does not, on the basis of available information, seem to result from the failure of a single reproductive pathway gene, but rather from an epistatic, possibly silencing, action exerted on the normal sexual reproductive pathway by a set of genes inherited as a unit and evolved in polyploid plants (Ozias-Akins et al., 1998
The high polyploidy and contrasting mode of reproduction of Poa pratensis should make it a model species for investigating apomixis. Sexually, P. pratensis reproduces through out-crossing or selfing, whereas, apomictically, it is a pseudogamous aposporic parthenogenetic species. The combination of a pollen recognition system and the aposporic nature of apomixis confers a strong ability to hybridize with, and retain, alien genomes (Wedin and Huff, 1996
We (Albertini et al., 2004 We now report the isolation and characterization of two genes, starting from the expressed sequence tag (EST) clones previously isolated in P. pratensis. Their genomic organization and characterization through temporal and spatial expression analysis of transcripts in reproductive tissues are reported. The putative involvement of these two genes in the process of ovule development and somatic embryo induction is also reported and discussed.
Cloning of Differentially Expressed Genes in Apomictically and Sexually Reproducing P. pratensis Genotypes
A differential display of mRNAs that combines cDNA-AFLP and developmentally staged inflorescences (Fig. 1) allowed Albertini et al. (2004)
Clone number 64 was 200 nucleotides in length and showed high similarity (E-value 9e-29, 83% identity) with an EST (accession no. BM084049) isolated from a pistil-specific cDNA library of apomictic Pennisetum ciliare (Jessup et al., 2003 ). It also showed high similarity (E-value 2e-44, 89% identity, 93% positivity) with the lipid-binding START domain-containing protein from Arabidopsis (accession no. NP193639). This P. pratensis cDNA fragment was named APOSTART and was chosen for further molecular characterization since its homolog of Arabidopsis (locus At4g19040) is located next to an unknown gene (locus At4g19050) that is strongly similar to a MOB1-like gene shown to be putatively involved in 2n egg formation in an apomeiotic mutant of M. falcata (Barcaccia et al., 2001). In Arabidopsis, both APOSTART-like and MOB1-like genes are also located on chromosome 5 in inverted position (loci At5g45560 and At5g45550, respectively).
Forward and reverse primers were designed for both 5'- and 3'-RACE. Several RACE experiments were required to obtain the entire 5'-end of both PpSERK and APOSTART. RACE identified two members of PpSERK and two of APOSTART. On the basis of full lengths, amplification of cDNA and genomic DNA samples with PpSERK1, PpSERK2, APOSTART1, and APOSTART2 specific primers enabled us to perform the end-to-end PCR and obtain complete cDNA and DNA clones of each allele. Allele specificity was confirmed in replicated experiments by directly sequencing the amplified products.
PpSERK1 mRNA sequence is 2,206 nucleotides in length and contains an open reading frame of 1,890 nucleotides, which encodes for a protein of 629 amino acids, and a 3'-untranslated region (UTR) of 316 nucleotides (accession no. AJ841698). Genomic sequencing of PpSERK1 showed that it is 5,188 nucleotides in length and contains 11 exons and 10 introns with a conserved exon/intron structure (supplemental data) identified in other SERK genes (Nolan et al., 2003 PpSERK1 and PpSERK2 amino acid sequence comparison performed with Vector AlignX software resulted in 97.0% identity and 98.1% positivity. In particular, 12 residues with different, and seven with similar, properties were scored. The overall nucleotide alignment scored 92.1% identity. The CDSs differed for 49 residues (97.4% identity), whereas the 3'-UTRs showed only 66.1% identity due not only to single residues but also to three indel regions. The final length of the genomic clone for APOSTART1 was 5,475 nucleotides and its sequence alignment with the relative full-length cDNA disclosed 21 introns (accession no. AJ786392). The 22 exons of APOSTART1 gave rise to a 2,187-nucleotide CDS encoding for a 728-amino acid protein (accession no. AJ786392). The APOSTART2 genomic clone was 5,441 nucleotides and, like APOSTART1, contained 21 introns and 22 exons for a 2,187-nucleotide CDS codifying for a 728-amino acid protein (accession no. AJ786393). The intron/exon structure showed to be conserved between species when compared with Arabidopsis and rice (see supplemental data). APOSTART1 and APOSTART2 amino acid sequence alignment resulted in 98.2% identity and 98.5% positivity. In particular, 10 residues with different, and three with similar, properties were scored along the 728-amino acid alignment.
Bioinformatic characterization of isolated cDNAs was carried out using the predicted proteins obtained from the full-length sequences. Using PpSERK as query enabled SERK-like proteins to be detected not only in Arabidopsis, rice, Medicago truncatula, and maize, but also in other less-known plant genomes like Daucus carota, Citrus unshiu, Ipomoea batata, and Gossypium hirsutum. In particular, PpSERK1 protein alignments showed it was very similar (E-value 0.0, 88% identity, 93% positivity) to the putative SERK1 protein from rice (accession no. BAD27594). It also showed 83% identity and 77% positivity (E-value of 0.0) with the Leu-rich repeat (LRR) transmembrane protein kinase of Arabidopsis (accession no. NP199390). Moreover, amino acid identity was 64% with GhSERK1-like, 46.1% with ZmSERK1, 45.4% with MtSERK1, 45.3% with AtSERK1, and 40.4% with DcSERK (accession nos. AAT64017, CAC37638, AAN64293, CAB42254, and AAB61708, respectively). It is worth noting that up to seven structurally distinct members were found in Arabidopsis. Two completely differentiated members were also found in rice. Therefore, plant genomes appear to contain multiple types of SERK genes. BLAST analysis revealed high similarity (E-value 0.0, 88% identity, 93% positivity) between PpSERK2 and the putative SERK1 protein from rice (accession no. BAD27594). It also showed 53% identity and 70% positivity (E-value of e-180) with the putative SERK located on chromosome 3 (locus At3g25560) of Arabidopsis (accession no. AAL91629). The alignment between PpSERK2 and SERK1-like proteins from other species resulted in values comparable to those obtained for PpSERK1. Multiple sequence alignments of a plant SERK protein subset were used to perform a phylogenetic analysis and obtain the consensus tree from the substitution matrix (Fig. 2). Differences in amino acid composition were consistent, mainly due to single substitutions and multiple residue gaps. Ordination analysis of both characterized SERK and SERK-like proteins according to their sequence similarity distinguished two main subgroups for plants. Figure 3 shows the amino acid alignment of PpSERK-like proteins mostly similar to PpSERK1 and PpSERK2.
Alignment of both PpSERK1 and PpSERK2 revealed the LRR domain (E-value 1e-07, score 52.3 bits, 25.6% aligned) and the Tyr kinase, catalytic (TyrKc) domain (E-value 9e-41, score 162 bits, 74.7% aligned). LRR domain-containing proteins are short sequences involved in protein-protein interactions, but the function of the LRR domain is still unknown, whereas the Tyr kinase is a phosphotransferase of the Tyr-specific kinase subfamily. Enzymes with TyrKc domains belong to an extensive family of proteins that share a conserved catalytic core common to both Ser/Thr and Tyr protein kinases. Database searches revealed a strong similarity (E-value 0.0, 86% identity, 90% positivity) between APOSTART1 and an unknown protein from rice (accession no. XP463792). It also showed that APOSTART1 was very similar (E-value 0.0, 65% identity, 79% positivity) to the pleckstrin homology (PH) domain-containing protein/lipid-binding START domain-containing protein (accession no. NP193639) from Arabidopsis (Fig. 4). Alignment revealed the presence of three conserved domains (see supplemental data): PH (E-value 4e-04, score 41.3 bits, 91.3% aligned), START (E-value 3e-28, score 121 bits, 87.7% aligned), and DUF1336 (E-value 2e-56, score 214 bits, 100% aligned).
The PH domain is commonly found in eukaryotic signaling proteins. The domain family possesses multiple functions, including the ability to bind inositol phosphates and various proteins. PH domains have been found to possess inserted domains (such as in phospholipase C-  syntrophins) and to be inserted within other domains. START domains are 200 to 210 amino acids in length and occur in proteins involved in lipid transport (phosphatidylcholine) and metabolism, signal transduction, and transcriptional regulation. The most striking feature of the START domain structure is a predominantly hydrophobic tunnel that extends for nearly the entire length of the protein and is used for binding a single molecule of large lipophilic compounds, for example, cholesterol. The DUF1336 domain is the C terminus of approximately 250 residues in a number of hypothetical plant proteins of unknown function. Although APOSTART2 and APOSTART1 are the same length, BLAST analysis revealed only two domains for APOSTART2: START (E-value 3e-27, score 118 bits, 87.7% aligned) and DUF1336 (E-value 1e-56, score 215 bits, 100% aligned). Even though the best amino acid alignment for APOSTART2 protein was detected in the same proteins of APOSTART1 (E-value 4e-74, 28% identity, 46% similarity), APOSTART2 also shared features with both an unknown protein (locus At5g35180) of Arabidopsis (accession no. BAA98203; E-value 1e-82, 31% identity, 48% similarity) and with an unknown protein of rice (accession no. BAD09877). This is important because, like APOSTART2, both the Arabidopsis and rice proteins (accessions BAA98203 and BAD09877) lack the PH domain. PSORT (version 6.4) analysis for plant proteins predicted the putative localization of both APOSTART1 (87.4% of probability) and APOSTART2 (86.6% probability) to the inner membrane of the mitochondrion.
Southern-blot hybridization analysis was performed to investigate the genomic organization of PpSERK and APOSTART. Total genomic DNAs were extracted from apomictic, sexual, and recombinant genotypes and digested with EcoRI, HindIII, and BamHI restriction enzymes. A 250-bp-long probe was obtained from genomic DNA amplification with specific primers corresponding to the C-terminal domain of PpSERK. The hybridization on genomic DNA digested with EcoRI resulted in six bands for the apomictic, six for the recombinant, and four for the sexual genotypes (Fig. 5A). Five hybridization products were found for genomic DNA of apomictic, four for the recombinant, and six for the sexual genotype when digested with HindIII. As high as eight hybridization products were found for the apomictic and recombinant genotypes, whereas only seven fragments were retrieved for sexual genotypes when genomic DNAs were digested with BamHI. Intriguingly, all three enzymes revealed that apomictic, sexual, and recombinant genotypes differed for several polymorphisms (Fig. 5A).
Specific primers for APOSTART were designed and produced a genomic fragment of 280 bp, which was used as a probe. As shown in Figure 5B, there were several hybridizing products in each digestion. APOSTART hybridization patterns suggest the presence of at least nine alleles/members in apomictic genotypes (HindIII digestion), eight in recombinant genotypes (HindIII digestion), and seven for sexual genotypes (EcoRI and HindIII digestion). As for PpSERK1, all three enzymes revealed that apomictic, sexual, and recombinant genotypes differed (Fig. 5B). The presence of multiple copies of APOSTART and SERK genes in the P. pratensis genome agrees with the finding of multiple members documented in genomes of other species.
Expression of PpSERK and APOSTART was assayed in genotypes with different modes of reproduction by using allele-specific primers in the reverse transcription (RT)-PCR analyses. Allele specificity was verified by directly sequencing an aliquot of the amplified products of each experiment. Reactions were performed in triplicate on independently isolated and retrotranscribed mRNAs from three apomictic, three sexual, and two recombinant genotypes and differences in quantitative data were tested for significance using ANOVA. The expression of a
Both PpSERK1 and PpSERK2 expression were relatively high in leaves and low in roots (Fig. 6, A–F). The expression pattern of two alleles was almost identical in apomictic and recombinant genotypes, but differed markedly in the sexual genotypes. Whereas in sexual genotypes PpSERK1 expression was high during premeiosis and decreased during meiosis and postmeiosis (Fig. 6B), it was modulated in the apomictic and recombinant genotypes (Fig. 6, A and C). In the last two, it was high in premeiosis, dropped during meiosis, then rose again to a level comparable to that recorded by sexual genotypes (Fig. 6, A–C). PpSERK2 expression rose from premeiosis to anthesis (Fig. 6E) in sexual genotypes, while in the apomictic and recombinant genotypes expression in inflorescences was higher in premeiosis than at any other stage (Fig. 6, D and F). In particular, expression dropped to a low level during meiosis, then, although it increased during postmeiosis and anthesis, it did not reach the premeiosis level (Fig. 6, D and F). APOSTART transcripts were visualized in inflorescences of apomictic, sexual, and recombinant genotypes, but no expression was detected in leaves or root tissues (Fig. 6, G–L). The APOSTART1 profile failed to differentiate between genotypes with different reproductive behavior. However, closer inspection of the quantitative data showed that APOSTART1 expression was higher in sexual and recombinant genotypes (17-fold and 10-fold, respectively) than in apomictic genotypes (Fig. 6, G–I). APOSTART2 was expressed at a low level in sexual genotypes during premeiosis and meiosis, but strongly during postmeiosis and anthesis (Fig. 6K). The expression pathway of the apomictic and recombinant genotypes was similar (Fig. 6, J and L). However, although the gene was expressed at a low level during premeiosis, it was not detected during meiosis, even when the number of amplification cycles was high (Fig. 6, J and L).
The spatial distribution of the PpSERK and APOSTART transcripts within reproducing organs of apomictic and sexual genotypes of P. pratensis was determined by a high number of independent in situ hybridization (ISH) experiments using digoxigenin (DIG)-labeled probes (see supplemental data). It should, however, be noted that, because of the very high sequence similarities between PpSERK1 and PpSERK2 and between APOSTART1 and APOSTART2, the probes used for ISH experiments could not distinguish alleles/members. In order to identify the putative role of PpSERK members in apomictic reproduction, analysis of their transcripts was limited to the first stages of ovule development. A partial PpSERK cDNA fragment containing a portion of the LRR and of the TyrKc domains and the entire region comprised between them was used as a PpSERK-specific probe. Results of the ISH experiments are given in Figure 7. Expression of PpSERK was first detected in the whole ovule primordium but not in other flower tissues (data not shown). A strong signal was detected during MMC differentiation in sexual genotypes (Fig. 7A). Interestingly, PpSERK transcripts were not detected in the MMC of apomictic flowers, whereas strong signals were found in some neighboring nucellar cells in all analyzed sections (Fig. 7B; see supplemental data).
Because APOSTART is an uncharacterized gene, its cellular expression was investigated in both anthers and ovaries. Ovaries were checked from the ovule primordium stage to mature embryo sac. Hybridization signals were recorded in both male and female reproductive cells for APOSTART (Fig. 7, D, E, G, and I), but no signals were detected in the sense slides (Fig. 7, F, H, and J). In particular, strong signals were detected in the megaspores during meiosis (Fig. 7, D and E) in all nucellar cells, as well as in the embryo sac at anthesis (Fig. 7G) in both apomictic and sexual genotypes, although the expression was higher in apomictic than sexual ones. Moreover, the signal was strong within anthers from pollen mother cells to the tetrad stage (Fig. 7I).
The Expression Pattern of PpSERK Is Compatible with Its Having a Role in the Specification of Aposporous Initials
The amino acid sequence of one of the 179 fragments (EST no. 6) was very like that of the SERK gene in Arabidopsis (At3g25560). This gene was named PpSERK. SERK is the only gene known to be implicated in the acquisition of embryogenic competence in plant cells. It was first isolated in carrot (Schmidt et al., 1997 We have cloned two SERK genes (PpSERK1 and PpSERK2) from the eight putative members derived from Southern hybridization analysis; both were expressed in sexual, apomictic, and recombinant genotypes, but their expression and/or activation/inactivation timing differed.
In apospory, a cell of the nucellus becomes an aposporous initial and then develops into a nonreduced embryo sac which, through parthenogenesis, gives rise to a viable embryo. How and why somatic cells of the ovule change their developmental fate and gain embryogenic potency is not known (Fehér et al., 2003 Our ISH data revealed that PpSERK is expressed in the MMC of sexual genotypes, but not in that of apomictic plants. In contrast, the strong signals detected in nucellar cells neighboring MMC suggest that PpSERK is involved in embryo sac development from nucellar cells.
EST number 64 was almost identical to an EST isolated from a pistil-specific cDNA library of apomictic P. ciliare (Jessup et al., 2003 The finding that APOSTART2 lacks the PH domain agrees with evidence that there is a single START-DUF1336 gene in both Arabidopsis and rice. Despite BLAST showing that APOSTART2 was most similar to the PH-START-DUF1336 gene of Arabidopsis, differences in the N-terminal amino acids typical of the PH domain were the same as those found in the members of Arabidopsis (At5g35180) and rice that lacked PH. Whether the lack of APOSTART2 during meiosis in both apomictic and recombinant genotypes has any effect on apomictic reproduction requires more extensive investigation. ISH analysis revealed that APOSTART members are expressed during both male and female meiosis in all micro- and megaspores. Moreover, strong signals were recorded up to the mature embryo sac stage.
Overall these data suggest that, in P. pratensis, APOSTART expression may also be related to the programmed cell death that is involved in the nonfunctional megaspore and nucellar cell degeneration events that permit enlargement of maturing embryo sacs (Wu and Cheung, 2000
The two genes isolated in this study, as well as others reported previously (Albertini et al., 2004 A detailed analysis of these genes and their specific ligands/substrates could provide us with a tool that would enable us to crack the code of apomixis.
Plant Material
A segregating F1 population of 68 plants was produced by crossing a completely sexual clone (S1/1-7, nonaposporic and nonparthenogenetic) with a highly apomictic genotype (RS7-3, aposporic and parthenogenetic). The sexual clone was derived from a cross between two completely sexual genotypes selected from German cultivars (Matzk, 1991
Cytological investigations showed that the chromosome number of parental genotypes was 2n = 36 for S1/1-7 and 2n = 64 for RS7-3. All progeny plants investigated were sired by agamospermous pollen and had a complement of 50 chromosomes (Porceddu et al., 2002
Florets of an apomictic, a sexual, and a recombinant genotype of Poa pratensis, were harvested and classified into four developmental stages (premeiosis, meiosis, postmeiosis, and anthesis), according to cytohistological investigations. Nucleic acids were isolated from about 0.5 g of fresh tissue (inflorescences at different developmental stages, leaves, and roots) using the GenElute mammalian total RNA miniprep kit (Sigma, St. Louis), according to the manufacturer's instructions, with some modifications to adapt it to plants. Total RNA was purified from residual genomic DNA by using the DNA-free (Ambion, Austin, TX) and the mRNA poly(A)+ purified by using the GenElute mRNA miniprep kit (Sigma). RT and second-strand synthesis was carried out with 1 µg of mRNA polyA+ and the standard procedure followed (Sambrook and Russell, 2001
Similarities for all cDNA-amplified fragments were searched in the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov) database using the 2.2.9 release with BLASTN (est_others database), BLASTX, and BLASTP (nr database) applications (Altschul et al., 1990
Clone-specific primers were used for performing both 5'- and 3'-RACE to obtain the full-length genes. The SMART RACE cDNA amplification kit (BD Biosciences Clontech, Palo Alto, CA) was applied to the mRNA poly(A)+ of the stage where the cDNA was scored, according to the manufacturer's instructions. Eight colonies for each RACE experiment were sequenced and full-length cDNA sequences were reconstructed from RACE fragments using VECTOR NTI Suite 8 Contig Express (InforMax).
Clone-specific primers were used for performing end-to-end amplifications of genomic DNA to obtain the entire transcriptional unit. An 0.7-µL aliquot of PCR-derived products was sticky-end ligated into a pCR4-TOPO vector using the TOPO TA cloning kit for sequencing (Invitrogen). The plasmid DNA was purified from 5 mL of an overnight culture on Luria-Bertani medium of Escherichia coli using the GenElute plasmid miniprep kit (Sigma). After a first confirmation sequence performed using M13 forward and reverse as primers, plasmids were used with the GeneJumper primer insertion kit for sequencing (Invitrogen). Sequences were then performed using the transposon-specific primers included in the kit. Full-length sequences were then obtained using Contig Express software (Invitrogen). Alignment between the full-length cDNAs and genomic clones disclosed the intron/exon structures of the genes.
For Southern analysis, 12 µg of P. pratensis genomic DNA (isolated with the Sigma GenElute plant genomic DNA kit) were digested with 60 units of EcoRI, HindIII, and BamHI, respectively (New England Biolabs, Beverly, MA). Restriction fragments were resolved by electrophoresis in a 0.8% agarose gel, blotted by capillary transfer, and linked at 80°C for 2 h to a Nitran N nylon membrane (Schleicher & Schull, Keene, NH). About 4 ng of each selected DNA or cDNA probe were labeled by PCR, using specific primers, in the presence of 32P-dCTP. Prehybridization, hybridization, and posthybridization washing of DNA membranes were performed according to Sambrook and Russell (2001)
First-strand cDNA was synthesized from 1 µg of P. pratensis total RNA in a 50-µL volume (Sambrook and Russell, 2001
The amplified PCR-generated products were directly sequenced to verify the allele specificity of primer pairs used and then separated on 2% agarose gel, blotted by capillary transfer, and linked at 80°C for 2 h to a Nitran N nylon membrane (Schleicher & Schull). The 32P-labeled probes were synthesized by PCR from purified PCR fragments. Filters were hybridized as in Vicient et al., (1999)
During inflorescence development, in sexual and apomictic P. pratensis genotypes, we distinguished four different stages (premeiosis, meiosis, postmeiosis, and anthesis). At each stage, single spikelets were collected, fixed in ethanol-formaldehyde-acetic acid, embedded in paraffin, and used for ISH experiments. Tissue preparation and hybridization conditions were the same as described by Angenent et al. (1995) Sense and antisense probes were obtained by in vitro transcription using cloned PCR-derived fragments of PpSERK1 and APOSTART as templates. In particular, PpSERK1 riboprobes were synthesized from a 1,110-bp fragment obtained using internal primers designed in a way so that the fragment contained small parts of the LRR and of the TyrKc domains and the entire region comprised between them. Moreover, APOSTART riboprobes were obtained from a single cDNA fragment of 1,060 bp, which comprised only a small part of the START and DUF1336 domains and the sequence between the two domains. Both PpSERK and APOSTART riboprobes did not discriminate between alleles due to the very small differences in sequences.
DIG-UTP sense and antisense riboprobes were synthesized by the T3 and T7 RNA polymerase. Transcripts were partially hydrolyzed by incubation at 60°C in 0.2 M Na2CO3/NaHCO3 buffer, pH 10.2, for about 35 min. Immunological detection was performed as described by Cañas et al. (1994) The genomic DNA and mRNA nucleotide sequences and the deduced amino acid sequences of the isolated PpSERK and APOSTART genes have been recorded. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AJ841698, AJ841697, AJ786392, and AJ786393.
We wish to thank Prof. Chris Gehring for providing Genevestigator data, Dr. Stefano Capomaccio for helping to name APOSTART and giving advice on the artwork, Dr. Luigi Russi and Prof. Peggy Ozias-Akins for critical reading of the manuscript, both Dr. Luca Pallottini and Dr. Lorenzo Raggi for their technical help, and Judy Etherington for her invaluable help with the English editing of the manuscript. Received February 28, 2005; returned for revision April 22, 2005; accepted May 8, 2005.
1 This work was supported by the Ministry of University and Research (project "Genetic aspects of seed production: an integrated approach towards the understanding of apomixis"). 
2 These authors contributed equally to the paper.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062059. * Corresponding author; e-mail emidalb{at}unipg.it; fax 39–075–585–6224.
Albertini E, Marconi G, Barcaccia G, Raggi L, Falcinelli M (2004) Isolation of candidate genes in Poa pratensis L. Plant Mol Biol 56: 879–894[Medline] Albertini E, Porceddu A, Ferranti F, Reale L, Barcaccia G, Romano B, Falcinelli M (2001) Apospory and parthenogenesis may be uncoupled in Poa pratensis: a cytological investigation. Sex Plant Reprod 14: 213–217 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410[CrossRef][ISI][Medline]
Altschul SF, Lipman DJ (1990) Protein database searches for multiple alignments. Proc Natl Acad Sci USA 87: 5509–5513
Altschul SF, Madden TL, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402 Angenent GC, Franken J, Busscher M, van Dijken A, van Went JL, Dons HJM, van Tunen AJ (1995) A novel class of MADS box genes is involved in ovule development in petunia. Plant Cell 7: 1569–1582[Abstract] Asker SE, Jerling L (1992) Apomixis in Plants. CRC Press, London Barcaccia G, Mazzucato A, Albertini E, Zethof J, Gerats T, Pezzotti M, Falcinelli M (1998) Inheritance of parthenogenesis in Poa pratensis L.: auxin test and AFLP linkage analysis support monogenic control. Theor Appl Genet 97: 74–82[CrossRef] Barcaccia G, Varotto S, Meneghetti S, Albertini E, Porceddu A, Parrini P, Lucchin M (2001) Analysis of gene expression during flowering in apomeiotic mutants of Medicago spp: cloning of ESTs and candidate genes for 2n eggs. Sex Plant Reprod 14: 233–238 Baudino S, Hansen S, Brettschneider R, Hecht VF, Dresselhaus T, Lorz H, Dumas C, Rogowsky PM (2001) Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family. Planta 213: 1–10[CrossRef][ISI][Medline] Bell G (1982) The Masterpiece of Nature. The Evolution and Genetics of Sexuality. University of California Press, Berkeley and Los Angeles, CA Bicknell RA, Koltunow AM (2004) Understanding apomixis: recent advances and remaining conundrums. Plant Cell 16: 228–245 Cañas LA, Busscher M, Angenent GC, Beltrán J, van Tunen AJ (1994) Nuclear localization of the petunia MADS box protein FBP1. Plant J 6: 597–604[CrossRef] Chaudhury AM, Peacock JW (1993) Approaches to isolating apomictic mutants in Arabidopsis thaliana: Prospects and progress. In GS Khush, ed, Apomixis: Exploiting Hybrid Vigor in Rice. International Rice Research Institute, Manila, The Philippines, pp 66–71 Chen LZ, Miyazaki C, Kojima A, Saito A, Adachi T (1999) Isolation and characterization of a gene expressed during early embryo sac development in apomictic guinea grass (Panicum maximum). J Plant Physiol 154: 55–62 Christenson LK, Strauss JF (2001) Steroidogenic acute regulatory protein: an update on its regulation and mechanism of action. Arch Med Res 32: 576–586[CrossRef][ISI][Medline] Desper R, Gascuel O (2002) Fast and accurate phylogeny reconstruction algorithms based on the minimum-evolution principle. J Comput Biol 9: 687–705[CrossRef][ISI][Medline] Dusi DMA (2001) Apomixis in Brachiaria decumbens Stapf. PhD Thesis, University of Wageningen, The Netherlands Fehér A, Pasternak TP, Dudits D (2003) Transition of somatic cells to an embryogenic state. Plant Cell Tissue Organ Cult 74: 201–228[CrossRef][ISI] Gamas P, de Billy F, Truchet G (1998) Symbiosis-specific expression of two Medicago truncatula nodulin genes, MtN1 and MtN13, encoding products homologous to plant defense proteins. Mol Plant Microbe Interact 11: 393–403[ISI][Medline] Gartner A, Milstein S, Ahmed S, Hodgkin J, Hengartner MO (2000) A conserved checkpoint pathway mediates DNA damage-induced apoptosis and cell cycle arrest in C. elegans. Mol Cell 5: 435–443[CrossRef][ISI][Medline] Grimanelli D, Leblanc O, Perotti E, Grossniklaus U (2001) Developmental genetics of gametophytic apomixis. Trends Genet 17: 597–604[CrossRef][ISI][Medline] Grossniklaus U, Schneitz K (1998) The molecular and genetic basis of ovule and megagametophyte development. Semin Cell Dev Biol 9: 227–238[CrossRef][ISI][Medline]
Hecht V, Vielle-Calzada JP, Hartog MV, Schmidt EDL, Boutilier K, Grossniklaus U, de Vries SC (2001) The Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 gene is expressed in developing ovules and embryos and enhances embryogenic competence in culture. Plant Physiol 127: 803–816 Jessup RW, Burson BL, Burow G, Wang YW, Chang C, Li Z, Paterson AH, Hussey MA (2003) Segmental allotetraploidy and allelic interactions in buffelgrass (Pennisetum ciliare (L) Link syn Cenchrus cili |
TOP
ABSTRACT
RESULTS
-tubulin allele was used as a reference for data standardization. 
