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First published online June 13, 2003; 10.1105/tpc.011742 American Society of Plant Biologists Sexual and Apomictic Reproduction in Hieracium subgenus Pilosella Are Closely Interrelated Developmental Pathways
a Department of Plant Science, Waite Campus, Adelaide University, Glen Osmond, South Australia 5064, Australia 4 To whom correspondence should be addressed. E-mail anna.koltunow{at}csiro.au; fax 61-8-83038601
Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in Hieracium.
Seed formation in angiosperms begins with the formation of the flower and the male and female reproductive organs, the anther and ovule, respectively. Each contains cells that undergo meiotic reduction and then mitosis to form the male and female gametophytes. The entry of two male sperm cells into the female gametophyte (embryo sac) and their respective fusion with the egg and two central cell nuclei therein during double fertilization enables the growth of the embryo and the endosperm compartments of the seed (Figure 1).
The events of sexual seed formation are characterized by spatial and temporal changes in the nuclear DNA content of cells with regard to both chromosome ploidy and the DNA contributed from maternal and paternal genomes. The gametophyte cell nuclei are reduced in DNA content, distinguishing them from the surrounding cells of the sporophyte. Fertilization restores the nuclear DNA content in the egg cell to that of the sporophytic generation, and each subsequent embryo cell contains maternal and paternal genomes present in equal ratio. By contrast, primary endosperm nuclei contain an unequal maternal-to-paternal genome ratio of 2:1, which is essential for seed viability in many species (Johnston et al., 1980  ; Lin, 1984; Chaudhury et al., 2001).
In some flowering plants, seeds also form asexually by apomixis. This term describes a highly variant suite of reproductive processes in which meiosis is avoided during embryo sac formation, embryo development is fertilization independent, and endosperm formation may or may not require fertilization (Nogler, 1984
The nature of the cells that initiate apomixis and the subsequent structures formed has been surmised from their positions within a tissue and their morphological appearance compared with those involved in sexual reproduction. Structures formed during apomixis can be similar to or can vary significantly in form and position relative to the surmised sexual counterpart (Nogler, 1984
Plants that reproduce by apomixis also retain the capacity to reproduce sexually to varying degrees. Sexual and apomictic reproduction appear independent, but they are not mutually exclusive. For example, in some apomicts, such as Hieracium (Figure 1), the sexual process ceases if apomixis initiates in the ovule, whereas in others, both processes occur side by side in a competitive manner. The coexistence of apomixis and sexual reproduction in an apomictic plant and the polyploid nature of most apomicts complicate genetic analyses. Nevertheless, it has been demonstrated that different modes of apomixis are conferred by a few Mendelian loci in which apomixis usually is dominant over sexual reproduction (do Valle and Savidan, 1996
However, there is a complete absence of data concerning the identity of cells that initiate apomixis, the molecular processes that regulate apomixis, and the relationship between sexual and apomictic pathways. Relatively few apomicts have been studied at the molecular level. Differential and subtractive hybridization have been attempted to isolate genes expressed during apomictic reproduction (Vielle-Calzada et al., 1996
Many apomicts and their sexual relatives are not readily amenable to plant transformation. This is not the case for composite Hieracium species, which display a generation time from seed to seed of 4 months and are suited to the molecular analysis of apomixis (Koltunow et al., 1995 Here, we examine the molecular relationships between sexual and apomictic pathways in Hieracium. Developmental markers from other plant species representing genes with known and informative expression patterns in sexual reproduction were introduced into sexual and apomictic plants. Comparative analyses of gene expression patterns showed that sexual and apomictic reproduction are molecularly related pathways that share regulatory programs.
AtFIS:GUS Gene Expression Marks Mature Embryo Sacs and Initiating Seed Structures in Sexual Hieracium Fertilization-independent (autonomous) endosperm development is one component of apomixis in Hieracium. In Arabidopsis thaliana (At), mutations in any of three members of the FERTILIZATION-INDEPENDENT SEED (FIS)–class genes MEDEA (MEA/FIS1), FIS2, and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE/FIS3) result in the fertilization-independent initiation of endosperm development (Ohad et al., 1996 ; Chaudhury et al., 1997; Grossniklaus et al., 1998). Chimeric AtMEA: -glucuronidase (GUS), AtFIS2:GUS, and AtFIE:GUS fusion constructs are coexpressed in comparable amounts during the late events of gametophyte development and early endosperm formation in transgenic Arabidopsis plants (Luo et al., 2000; Yadegari et al., 2000). However, the spatial expression patterns of the three chimeric genes vary slightly in developing seeds: AtFIS2:GUS activity is specific to the endosperm nuclei, AtMEA: GUS activity is restricted to the nuclear endosperm domains, and AtFIE:GUS activity is detected in both the endosperm nuclear domains and the embryo. The expression of AtFIS2:GUS was detected first around the two central cell nuclei before their fusion (Figure 2A) and continued in the dividing endosperm nuclei until the fifth or sixth nuclear division (Figure 2B) and thereafter only in the endosperm cyst (Figure 2C). AtFIE:GUS expression also is detected during pollen development (Luo et al., 2000).
The three chimeric AtFIS:GUS genes were introduced into Hieracium to compare their expression during fertilization-dependent and fertilization-independent seed development. The expression patterns of AtFIS2:GUS (Figures 2D to 2F) and AtMEA:GUS (data not shown) were identical at embryo sac maturity and during early seed development in sexual Hieracium P4. High levels of GUS activity were detected around the polar nuclei within the embryo sac and at a lower level throughout the central cell, the egg apparatus, and the endothelium (Figure 2D). After fertilization, AtFIS2:GUS expression in sexual Hieracium resembled that observed in Arabidopsis and was associated with the primary endosperm nucleus and the dividing endosperm nuclei (Figure 2E). However, AtFIS2:GUS expression was not localized tightly to endosperm nuclei, as observed in Arabidopsis (cf. Figures 2B and 2E), and continued to be expressed in endosperm cells after the formation of cell walls (Figure 2F). During embryogenesis, AtFIS2:GUS expression was detected in the dividing zygote and persisted until the heart stage of embryo development (Figures 2E and 2F). The same expression was observed for AtMEA:GUS in fertilized Hieracium embryos (data not shown), and this has not been observed in Arabidopsis. The expression of AtFIE:GUS at embryo sac maturity and during early endosperm and early embryo development was barely detectable in the transgenic plants examined (Figure 2N) compared with the expression of the AtMEA:GUS and AtFIS2:GUS genes. However, the intensity of AtFIE:GUS expression increased during the later stages of embryo development in Hieracium (Figure 2O), attaining a level comparable to that of the other two genes. The expression patterns of the three AtFIS:GUS genes at embryo sac maturity and seed initiation in sexual Hieracium were similar but not identical to those observed in Arabidopsis. However, the coordinated expression of the heterologous AtFIS:GUS genes in key structures formed during sexual reproduction underscored their suitability as markers for comparing gene expression patterns during aposporous embryo sac formation and fertilization-independent seed initiation in apomictic Hieracium.
Conservation of AtFIS:GUS Expression during Embryo Sac Maturation and Seed Initiation in Sexual and Apomictic Hieracium Like that observed in sexual P4, the expression patterns of the AtFIS2:GUS (Figures 2G to 2M) and AtMEA:GUS (data not shown) chimeric genes were identical during embryo sac and fertilization-independent seed development in apomictic Hieracium. GUS activity arising from the introduced AtFIS2:GUS gene was evident in mature aposporous embryo sacs situated in the micropylar region (Figure 2G), multiple coalescing embryo sacs in A3.4 (Figure 2J), and aposporous embryo sacs found in the chalazal region of ovules from the transgenic apomictic Hieracium plants (Figure 2K). Similar to the expression in sexual P4, AtFIE:GUS expression was significantly lower than that of the other two genes during early embryo sac development (data not shown). Just before the initiation of fertilization-independent endosperm in both apomicts, a high level of AtFIS2:GUS activity was detected around nuclei positioned in the center of the aposporous embryo sac, and a lower level of GUS activity was observed consistently throughout the central cell (Figure 2G). This increase in staining around the polar nuclei suggests that, as in sexual reproduction, central cell nuclei are the likely progenitors of autonomous endosperm in apomictic Hieracium. The nuclei formed during fertilization-independent endosperm divisions also were marked with AtFIS2:GUS (Figure 2H). After cell walls had formed between the syncytial nuclei, the endosperm cells continued to express the AtFIS2:GUS gene until the torpedo stage of embryo development (Figure 2I), consistent with the expression observed in the fertilized sexual plant (Figure 2F). Coexpression of AtFIS2:GUS and AtMEA:GUS also was observed in patches of endosperm that failed to cellularize (data not shown) and in the cellularized endosperm of seeds in which embryos failed to initiate (Figure 2M). The AtFIS2:GUS (Figures 2H, 2I, and 2L), AtMEA:GUS, and AtFIE:GUS (data not shown) genes were coexpressed during fertilization-independent embryo formation when an embryo formed at the micropylar end of the aposporous embryo sac (Figure 2I) and also in multiple embryos that formed in different spatial locations (Figures 2H and 2L). GUS activity from the three chimeric genes in apomictic seeds diminished at the mid to late heart stage of embryo development (Figure 2I), comparable to the expression in the sexual plant (cf. with Figure 2F). The three AtFIS:GUS genes were coexpressed in maturing aposporous embryo sacs and in the early embryo and endosperm of fertilization-independent seeds of both apomictic Hieracium species. The spatial and temporal expression patterns were comparable to those observed in embryo sacs, embryos, and endosperm in sexual Hieracium. This finding indicates that the spatial and temporal regulation of the introduced AtFIS:GUS genes is common to both sexual and apomictic pathways despite the absence of meiosis and fertilization in the apomictic pathway.
AtSERK1:GUS and SERK-Like Expression Confirm Shared Molecular Elements during Sexual and Apomictic Reproduction To determine if Hieracium contained SERK-like sequences, degenerate primers were designed to amplify part of the conserved kinase domain common to DcSERK and AtSERK1 (Figure 3A). Sequence analysis of the cloned reverse transcriptase–mediated (RT) PCR products obtained from early D3 Hieracium seeds revealed a partial 516-bp SERK-like clone (HpSERK-L) that was 82% identical to the DcSERK coding sequence. The cDNA fragment also showed >80% identity at the DNA level to the Arabidopsis SERK1, SERK2, and SERK3 genes. The HpSERK-L, DcSERK, and AtSERK1 putative amino acid sequences were almost identical across 7 of the 11 kinase domains (Figure 3B).
Hybridization of the HpSERK-L fragment to genomic DNA from sexual P4, apomictic D3, and apomictic A3.4 Hieracium revealed that few copies of the gene were likely to be present in these different polyploid plants (Figure 3C). Labeled AtSERK1 cDNA probe hybridized to the same genomic DNA fragments (data not shown). RT-PCR analysis using sequence-specific primers showed the presence of HpSERK-L mRNA transcripts from early ovule development until early seed development in both sexual and apomictic Hieracium (Figure 3D). Similar levels of expression were detected in both plants, and this temporal pattern of HpSERK-L gene expression corresponded to that observed for AtSERK1, except that HpSERK-L transcript also was detected in young, unexpanded Hieracium leaves. These data confirmed the presence of SERK-like sequences in Hieracium, and subsequent expression analyses used the AtSERK1:GUS and DcSERK probes with known expression patterns. When the AtSERK1:GUS gene was introduced into Hieracium, the same spatial and temporal pattern of expression was observed in both sexual and apomictic plants. GUS activity was detected initially in developing florets in the region destined to give rise to the inferior ovary (data not shown). As the MMC and megaspores developed in the ovule, the expression of AtSERK1:GUS was not restricted to the nucellar region containing the megaspores, which is the case in Arabidopsis, but GUS activity was observed throughout the ovule (Figure 4A).
AtSERK1:GUS expression was not detected during megagametogenesis in sexual P4 or during aposporous embryo sac formation in apomictic D3, but expression was restricted to cells surrounding the vascular bundle of the ovule (Figure 4B), a pattern not evident in Arabidopsis. AtSERK1:GUS activity was not detected clearly in the egg cell of sexual plants or within the cells of the aposporous embryo sacs at seed initiation, and expression may have been below the sensitivity limit of detection. However, after fertilization in sexual Hieracium, embryos expressed GUS from the early four- to eight-cell stage until the heart stage of embryogenesis (Figure 4C). AtSERK1:GUS expression was not observed in developing endosperm. Similarly, in apomictic Hieracium, globular and also irregular clumps of cells morphologically resembling embryos were marked with GUS activity (Figure 4D), and embryo expression of AtSERK1:GUS began to decrease soon after the transition to the heart stage of development. DcSERK was used as an antisense probe in whole-mount in situ hybridization (WISH) experiments. The same pattern of transcript localization was observed in both sexual and apomictic Hieracium and was essentially identical to that observed in transgenic plants expressing the AtSERK1:GUS gene. SERK-like mRNA sequences were detected in young ovules (Figure 4E) but not in the egg cell or the zygotes of sexual plants. WISH analysis of seeds from diploid apomictic Hieracium D2 that display a high frequency of multiple fertilization-independent embryos failed to detect SERK-like transcript until the embryos contained at least four to eight cells (Figure 4F). Similar to the AtSERK1:GUS expression, SERK-like mRNA sequences were detected clearly in small globular embryos (Figure 4G) and subsequently in embryos until the early heart stage in both sexual P4 (Figure 4C) and apomictic D2 (Figure 4H). These expression data reflect in part the patterns observed for AtSERK1 in Arabidopsis and suggest a role for endogenous Hieracium SERK-like sequences during ovule and seed development. Both the AtSERK1:GUS gene and an endogenous HpSERK-L gene appear to be regulated spatially and temporally in a similar manner in sexual and apomictic Hieracium. The conserved patterns of SERK and AtFIS:GUS marker expression during the temporal events of sexual and apomictic reproduction in Hieracium collectively support the conclusion that both sexual and apomictic pathways share common molecular regulatory features and are not distinct pathways.
Aposporous Initial Cells Do Not Express an MMC Marker
The marker genes available to test this are limited. The SPROCYTELESS (SPL) gene (Yang et al., 1999 In sexual Hieracium (P4) plants, AtSPL:GUS expression was localized specifically to the male and female sporogenic tissues. GUS activity was detected clearly in developing anthers in pollen mother cells (Figures 5A and 5B) and microspore tetrads (Figure 5C), and also in the ovule, where it was restricted to the MMC (Figure 5D). In contrast to the expression of AtSPL:GUS in Arabidopsis ovules, GUS activity in sexual Hieracium was not evident in the nucellar epidermis or in any of the megaspores during functional megaspore selection (Figure 5E) or degeneration. The MMC-specific pattern was observed in four independent lines by dark-field microscopy and Nomarksi differential interference contrast microscopy and verified by serial sections (data not shown). AtSPL:GUS activity was detected clearly in MMCs within ovules from five apomictic (D3) Hieracium lines (Figure 5F) but not in the aposporous initial cells that differentiated soon afterward when the MMC underwent meiosis (Figure 5G).
These data show that the expression of the AtSPL:GUS chimeric gene clearly marks MMCs in Hieracium ovules but, unlike in Arabidopsis, it does not mark later temporal events of megasporogenesis. Nevertheless, the expression patterns in Hieracium show that at the time of cytologically discernible differentiation, aposporous initial cells do not express the AtSPL:GUS MMC marker gene; hence, they are unlikely to share identity with the MMC.
AtFIS2:GUS First Marks Megaspores and Is Downregulated in the Selected Megaspore
In situ hybridization using a probe to detect GUS mRNA transcripts was performed to determine if the megaspore expression pattern in transgenic plants containing AtFIS2:GUS was related to the selective distribution of mRNA synthesized in the MMC before meiosis or to regulatory events that occur during and/or after meiosis. AtFIS2:GUS mRNA was not detectable in the MMC of sexual plants (Figure 7A). Transcripts were not detected in the selected functional megaspore after meiosis but were evident in degenerating megaspores and some of the degenerating nucellar cells (Figures 7B and 7C). Once the nucleus of the functional megaspore divided, AtFIS2:GUS mRNA was detected in the early embryo sac (data not shown).
The absence of both AtFIS2:GUS mRNA and protein activity in the enlarged functional megaspore was not a consequence of the absence of the AtFIS2:GUS gene from that cell as a result of meiotic segregation. Seven independent sexual AtFIS2:GUS transgenic lines were generated, and the copy number of the gene in these hemizygous plants varied from three to five insertions (Figure 7E). In AtFIS2:GUS plants containing a single copy of the transgene, 50% of the megaspores should contain the gene after meiosis, whereas all of the megaspores are likely to contain the transgene in plants with multiple unlinked copies of the gene. Examination of 657 ovules from sexual P4 plants with different numbers of transgene insertions showed that the temporal and spatial patterns of AtFIS2:GUS expression were surprisingly conserved between MMC differentiation and meiosis. GUS activity was never evident in the MMC, but in ovules that contained early megaspores soon after meiosis (n = 148), GUS activity was detected in all four spores, with the activity in the selected chalazal-most spore weaker than that of the other megaspores. Ovules that contained an enlarged functional megaspore displayed GUS activity only in the three degenerating megaspores (n = 509). This finding suggests that in plants containing multiple copies of the transgene, in which all meiotic products should inherit a copy, processes associated with functional megaspore specification also are involved in switching off AtFIS2:GUS expression in that cell.
AtFIS2:GUS Is Expressed Differentially at Meiosis in Apomictic Hieracium In both apomictic Hieracium species, AtFIS2:GUS expression was completely absent from aposporous initial cells as they enlarged and expanded toward the tetrad (Figures 6F and 6J), just as it was absent from the enlarging selected megaspore in sexual plants. In the apomict D3, a single aposporous initial usually expanded into the position vacated by the degenerating megaspores and nucellar epidermis (Figure 6F). GUS activity was absent from the aposporous initial cell as it expanded into the position vacated by the degenerated megaspores (Figure 6G), but it was detected in the aposporous structure as soon as the nucleus divided and aposporous embryo sac formation began (Figure 6H). In the apomict A3.4, multiple aposporous initials differentiated, and none of these initials expressed AtFIS2:GUS (Figure 6K) until each nucleus divided to initiate aposporous embryo sac development (Figure 6L). As in D3, the expression of AtFIS2: GUS in A3.4 ovules also was evident in the nucellar epidermis and in all four megaspores until these cells had degenerated. The reasons for the expression of GUS in all four meiotic products of apomictic plants containing only a single insert of the AtFIS2:GUS transgene (Figure 7E) are not clear. Repeated transformation experiments have failed to identify a single-copy sexual P4 AtFIS2:GUS line to enable comparison. We speculate that postmeiotic movement of either the mRNA or the protein may occur during megaspore and/or nucellar epidermal cell degeneration. The observation that AtFIS2:GUS expression is observed first in AI cells when the nucleus undergoes mitosis during early embryo sac formation suggests that the AI cell may acquire functional identity resembling that of a selected megaspore soon after differentiation. Alternatively, the AI cell may switch to an embryo sac program at the initiation of nuclear mitosis. These possibilities will require further clarification with appropriate markers as they become available.
Apomixis Reflects a Deregulated Sexual Program In the past, apomixis and sexual reproduction were viewed as two distinct processes that have little in common. Ernst (1918) postulated that apomixis might result from the hybridization of related species based on the observation that the apomicts examined at that time were polyploid and highly heterozygous. The pioneering studies of Nogler (1984) and Savidan (1982) proved that apomixis is under genetic control, although genetic modifiers or environmental conditions may affect its expressivity (Nogler, 1984; Savidan, 2000). The subsequent confirmation that relatively few loci control different modes of apomixis and that mutagenesis can induce apparent components of apomixis in sexual plants has lent further support to the hypothesis that apomixis and sexual reproduction might share common elements. The expression patterns of the AtSPL:GUS, AtSERK1:GUS, and various AtFIS-class:GUS chimeric constructs in sexual and apomictic Hieracium species described here have directly confirmed that apospory and the components of autonomous embryo and endosperm development share gene expression and regulatory components with sexual reproduction. This is in spite of the comparative structural differences in embryo sac, embryo, and endosperm development and also the temporal and spatial changes in cell and tissue ploidy status in the two reproductive pathways. The spatial and temporal expression data of the marker genes were integrated to devise a model for the control of apomixis in Hieracium (Figure 8). Expression of the chimeric constructs was shared from the first nuclear division of the AI cell and the selected megaspore and continued during the mitotic events of embryo sac formation and also fertilization-dependent and fertilization-independent embryo and endosperm formation in sexual plants and apomictic plants, respectively. The markers used did not confirm the functional identity of the AI cell during differentiation from the nucellus and its subsequent enlargement but showed that the AI cell was unlikely to share identity with the MMC during this sequence of events.
The results of marker analyses indicate that aspects of meiosis are avoided during aposporous embryo sac formation in Hieracium, because the AI cell that differentiates from the nucellus (or nucellar epidermis) is directed onto a mitotic embryo sac formation pathway combined with fertilization-independent embryo and endosperm development. This finding, coupled with the shared nature of gene expression programs and regulators in sexual and apomictic pathways, suggests that apomixis is manifested by the induction of a sexual developmental program deregulated in both time and space that leads to cell fate changes and the omission of critical steps in the sexual process (Figure 8).
Interactions between Sporophytic Tissues and Sexual and Apomictic Pathways
Differentiation of the AI cell in apomictic Hieracium coincides with the demise of the sexual process in the ovule. Previous studies have shown that this does not simply occur by physical displacement (Tucker et al., 2001
Molecular studies in Arabidopsis have suggested that positional signals involving the functional megaspore control cell fate and degeneration and result in the death of the three nonselected megaspores during megaspore selection in sexual plants (Wu and Cheung, 2000
Other aposporous species form initials at different times of ovule development compared with Hieracium species, and in some of these, such as Brachiaria species (Araujo et al., 2000
Roles of FIS-Class Genes and Genes That Stimulate Somatic Embryogenesis in Apomictic Reproduction
The early expression of AtFIS2:GUS in Hieracium and the conservation of synchronized expression of the AtMEA:GUS, AtFIS2:GUS, and AtFIE:GUS genes during the late events of embryo sac formation and early seed development raise questions concerning the role of Hieracium FIS-class genes during sexual and apomictic reproduction. The FIS-class genes are postulated to form complexes that alter chromatin structure to inhibit or promote the expression of seed development genes and to regulate endosperm polarity in Arabidopsis (Sorensen et al., 2001 A plausible hypothesis is that the altered regulation of Hieracium FIS genes may enable autonomous seed development in apomictic plants. We have isolated homologs of the Arabidopsis FIS genes from sexual and apomictic Hieracium species. Analysis of transgenic plants in which these genes have been upregulated and downregulated may determine their function during fertilized and autonomous seed development.
Plant Material All Hieracium plants examined were maintained by vegetative propagation under the growth conditions described by Koltunow et al. (1998) . Four different genetically characterized Hieracium lines that have been described previously (Koltunow et al., 1998, 2000; Tucker et al., 2001) were used in this study. P4 is a self-incompatible tetraploid sexual accession of H. pilosella, D3 is an apomictic triploid accession of H. piloselloides, D2 is derived from D3 and is an apomictic diploid derived from parthenogenetic development of a reduced egg cell, and A3.4 is an apomictic aneuploid accession of H. aurantiacum. In all three apomictic plants, >93% of the viable seeds are apomictic (Koltunow et al., 1998, 2000). Tissues for GUS staining, histology, and in situ hybridization were staged and collected as described by Koltunow et al. (1998).
Arabidopsis thaliana ecotype Landsberg erecta seeds homozygous for AtFIS2:GUS were provided by Abed Chaudhury (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia) and grown at 20°C with a 16-h daylength. Ovules and early seeds were staged as described by Schneitz et al. (1995)
Arabidopsis SPL, MEA, FIS2, FIE, and SERK1 Chimeric Genes and Hieracium Transformants
To determine transgene copy number, genomic DNA was isolated from the leaves of transformed plants as described by Tucker et al. (2001)
Isolation, Reverse Transcriptase–Mediated PCR Analysis, and Genomic Organization of a Hieracium SERK Homolog
RNA samples were harvested from various stages of leaf and ovary development (ovule initiation and heart stage of embryogenesis) (Koltunow et al., 1998
Genomic DNA was isolated from the leaves of P4, D3, and A3.4 plants as described by Tucker et al. (2001)
Whole-Mount in Situ Hybridization of a Hieracium SERK Homolog
In Situ Localization of GUS mRNA A 515-bp fragment of GUS (for primers, see above) was amplified by PCR from pBI101-3 and cloned into pGEM T-Easy (Promega). The clone was linearized with SalI (sense) and NcoI (antisense), and DIG-labeled RNA probe was produced using the DIG-RNA labeling kit (Boehringer Mannheim) and SP6 or T7 RNA polymerase to generate sense and antisense probes, respectively.
Microscopy and GUS Staining Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes.
This article is dedicated to the memory of our friend, Nicholas Paech, a student who contributed much to our work on apomixis and who passed away on September 25, 2002. We thank Venkatesan Sundaresan for the AtSPL:GUS construct, Abed Chaudhury and Ming Luo for the AtFIS: GUS constructs and Landsberg erecta AtFIS2:GUS seeds, Melissa Pickering for Arabidopsis GUS stains, and Anne Tassie for generating Hieracium transformants. We also thank Ueli Grossniklaus, Ross Bicknell, members of the European Plant Embryogenesis Network funded by European Union Framework 4, and members of the CSIRO Plant Industry Molecular Basis of Seed and Fruit Formation laboratory for many helpful discussions. This study was supported by an Adelaide University Premier Scholarship to M.R.T., an Australian Center for International Agricultural Research grant to A.M.G.K., a Brazilian Agricultural Research Corporation grant to A.-C.G.A., and a University of Amsterdam travel fellowship to J.-B.R.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.011742.
1 Current address: School of Plant Science, The University of Tasmania, Hobart, Tasmania 7001, Australia.
2 Current address: Gene Twister, P.O. Box 193, 6700AD Wageningen, The Netherlands.
3 Current address: School of Biochemistry and Molecular Biology, The Australian National University, Canberra, ACT 0200, Australia. Received March 9, 2003; accepted May 13, 2003.
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Abstract
INTRODUCTION
-32P-dCTP–labeled GUS probe (515-bp PCR fragment; see above). Hybridization solutions and washes were as described by Tucker et al. (2001)
25 ng) of cDNA, 0.5 µM of each gene-specific primer, 1 unit of RedTaq polymerase (Sigma), 2 µL of 10x PCR buffer (100 mM Tris-HCI, pH 8.3, 500 mM KCI, 11 mM MgCI2, and 0.1% gelatin) and 50 µM of each deoxynucleotide triphosphate. Primers used for the RT-PCR procedure were HpSERKF (5'-GGCTGTCAAAACGGGTTAAA-3') and HpSERKR (5'-CATTTGGTGGTCGCTCTCTTAA-3'). PCR samples were transferred to Hybond-N membranes (Amersham) and hybridized with 
