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Arabidopsis does not flower in winter because FLC represses flowering genes, but prolonged cold exposure silences FLC, allowing flowering in spring. How do plants recalibrate this switch to adapt to different climates? Reporting in Science, Coustham et al. (2012) found that tweaking a Polycomb target sequence may do the trick.

Plants can remember periods of cold several weeks later and respond by switching from vegetative to reproductive development. Recent findings shed light on this phenomenon by showing that a gene responsible for keeping this memory encodes a member of the Polycomb group of proteins.

In angiosperms, the sperm cells are carried within the pollen tubes (male gametophytes) to the female gametophyte so that double fertilization can occur. The female gametophyte exerts control over the male, with specialized cells known as synergids guiding the pollen tubes and controlling their behavior when they enter the female gametophyte so that the sperm cells can be delivered to the egg and central cell. Upon pollen tube arrival at the ovule, signal transduction cascades mediated by receptor-like kinases are initiated in both the synergid and the tip of the pollen tube, leading to synergid cell death and pollen tube rupture. In this review, we discuss the role of these receptors and of newly discovered members of the pollen tube reception pathway.

In plants, the male and female gametophytes represent the haploid generation that alternates with the diploid sporophytic generation. Male and female gametophytes develop from haploid micro- and megaspores, respectively. In flowering plants (angiosperms), the spores themselves arise from the sporophyte through meiotic divisions of sporogenous cells in the reproductive organs of the flower. Male and female gametophytes contain two pairs of gametes that participate in double fertilization, a distinctive feature of angiosperms. In this paper, we describe the employment of a transposon-based gene trap system to identify mutations affecting the gametophytic phase of the plant life cycle. Mutants affecting female gametogenesis were identified in a two-step screen for (i) reduced fertility (seed abortion or undeveloped ovules) and (ii) segregation ratio distortion. Non-functional female gametophytes do not initiate seed development, leading to semi-sterility such that causal or linked alleles are transmitted at reduced frequency to the progeny (non-Mendelian segregation). From a population of 2,511 transposants, we identified 54 lines with reduced seed set (2%). Examination of their distorted segregation ratios and seed phenotypes led to the isolation of 12 gametophytic mutants, six of which are described herein. Chromosomal sequences flanking the transposon insertions were identified and physically mapped onto the genome sequence of Arabidopsis thaliana. Surprisingly, the insertion sites were often associated with chromosomal rearrangements, making it difficult to assign the mutant phenotypes to a specific gene. The mutants were classified according to the process affected at the time of arrest, i.e. showing mitotic, karyogamic, maternal or degenerative phenotypes.

Background Epigenetic regulation of gene dosage by genomic imprinting of some autosomal genes facilitates normal reproductive development in both mammals and flowering plants. While many imprinted genes have been identified and intensively studied in mammals, smaller numbers have been characterized in flowering plants, mostly in Arabidopsis thaliana. Identification of additional imprinted loci in flowering plants by genome-wide screening for parent-of-origin specific uniparental expression in seed tissues will facilitate our understanding of the origins and functions of imprinted genes in flowering plants. Results cDNA-AFLP can detect allele-specific expression that is parent-of-origin dependent for expressed genes in which restriction site polymorphisms exist in the transcripts derived from each allele. Using a genome-wide cDNA-AFLP screen surveying allele-specific expression of 4500 transcript-derived fragments, we report the identification of 52 maternally expressed genes (MEGs) displaying parent-of-origin dependent expression patterns in Arabidopsis siliques containing F1 hybrid seeds (3, 4 and 5 days after pollination). We identified these MEGs by developing a bioinformatics tool (GenFrag) which can directly determine the identities of transcript-derived fragments from (i) their size and (ii) which selective nucleotides were added to the primers used to generate them. Hence, GenFrag facilitates increased throughput for genome-wide cDNA-AFLP fragment analyses. The 52 MEGs we identified were further filtered for high expression levels in the endosperm relative to the seed coat to identify the candidate genes most likely representing novel imprinted genes expressed in the endosperm of Arabidopsis thaliana. Expression in seed tissues of the three top-ranked candidate genes, ATCDC48, PDE120 and MS5-like, was confirmed by Laser-Capture Microdissection and qRT-PCR analysis. Maternal-specific expression of these genes in Arabidopsis thaliana F1 seeds was confirmed via allele-specific transcript analysis across a range of different accessions. Differentially methylated regions were identified adjacent to ATCDC48 and PDE120, which may represent candidate imprinting control regions. Finally, we demonstrate that expression levels of these three genes in vegetative tissues are MET1-dependent, while their uniparental maternal expression in the seed is not dependent on MET1. Conclusions Using a cDNA-AFLP transcriptome profiling approach, we have identified three genes, ATCDC48, PDE120 and MS5-like which represent novel maternally expressed imprinted genes in the Arabidopsis thaliana seed. The extent of overlap between our cDNA-AFLP screen for maternally expressed imprinted genes, and other screens for imprinted and endosperm-expressed genes is discussed., BMC Plant Biology, 11, ISSN:1471-2229

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate., Author Summary Germline specification is a key step in sexual reproduction. In plants, the reproductive lineage or “germline” doesn't arise early in development, as it does in animals; rather, the germline is specified during flower development. In the female reproductive organs of the flower, a single sporophytic cell in each ovule is selected and differentiates into a megaspore mother cell (MMC), which will undergo meiosis. Despite the importance of the specification of the MMC as the first committed cell of the germline lineage, little is known about the genetic and molecular bases of this process. We performed a cell-type-specific transcriptome analysis of Arabidopsis MMCs using laser-assisted microdissection and microarrays. Statistical data analysis comparing these results with the transcriptomes of 71 other types of cells and tissues revealed the importance of translational control pathways and RNA helicases for plant germline development, a feature reminiscent of the animal germline. We further characterized a novel MMC-enriched RNA helicase, called MNEME, and showed that it plays important roles in MMC differentiation and the restriction of the plant germline to only one cell per ovule. This example illustrates the usefulness of our transcriptome dataset for the identification of novel candidate genes involved in this crucial step of plant reproduction.

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development., Author Summary Understanding the convergent developmental mechanisms of core cell cycle genes is highly instructive in biology. When these genes are essential in development, lethality precludes mutation analysis throughout the life cycle of an organism. We subjected a homozygous lethal mutation in RETINOBLASTOMA RELATED (RBR) of Arabidopsis for tetraploid genetic analysis to study the function of RBR during the plant life cycle. In diploids, while RBR–deficient female gametophytes with features of aberrant cell fate and differentiation were analogous to what was previously reported for male gametophytes, we provide evidence that RBR controls gametic genome duplication, thus genome integrity in the gametophyte-derived progeny. Quantitative reduction of RBR in tetraploids led to identification of rbr heterozygous plants that displayed novel RBR dosage-dependent phenotypes in differentiation and development of the sporophyte albeit the absence of cell cycle defects. These phenotypes coincided with deregulation of conserved epigenetic factors such as Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the sporophyte, as shown for the gametophytes as well. However, unlike the repression by the PRC2 in gametophytes, RBR is activated by the sporophytic PRC2 subunits, suggesting that distinct modules of the conserved RBR-PRC2-MET1 loop control gametophyte and sporophyte generations in plants.

Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference–mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.

Competition and disturbance are potent ecological forces that shape evolutionary trajectories. These forces typically work in opposition: when disturbance is infrequent, densities are high and competition is intense. In contrast, frequent disturbance creates a low-density environment in which competition is weak and good dispersal essential. We exploited recent advances in genomic research to quantify the response to selection by these powerful ecological forces at the phenotypic and molecular genetic level in experimental landscapes. We grew the annual plant Arabidopsis thaliana in discrete patches embedded in a hostile matrix and varied the number and size of patches and the intensity of disturbance, by creating both static and dynamic landscapes. In static landscapes all patches were undisturbed, whereas in dynamic landscapes all patches were destroyed in each generation, forcing seeds to disperse to new locations. We measured the resulting changes in phenotypic, genetic, and genotypic diversity after five generations of selection. Simulations revealed that the observed loss of genetic diversity dwarfed that expected under drift, with dramatic diversity loss, particularly from dynamic landscapes. In line with ecological theory, static landscapes favored good competitors; however, competitive ability was linked to growth rate and not, as expected, to seed mass. In dynamic landscapes, there was strong selection for increased dispersal ability in the form of increased inflorescence height and reduced seed mass. The most competitive genotypes were almost eliminated from highly disturbed landscapes, raising concern over the impact of increased levels of human-induced disturbance in natural landscapes.

Much of our knowledge on heredity, development, physiology and the underlying cellular and molecular processes is derived from the studies of model, or reference, organisms. Despite the great variety of life, a common base of shared principles could be extracted by studying a few life forms, selected based on their amenability to experimental studies. Very briefly, the origins of a few model organisms are described, including E. coli, yeast, C. elegans, Drosophila, Xenopus, zebrafish, mouse, maize and Arabidopsis. These model organisms were chosen because of their importance and wide use, which made them systems of choice for genome-wide studies. Many of their genomes were between the first to be fully sequenced, opening unprecedented opportunities for large-scale transcriptomics and proteomics studies.

Embryogenesis is a critical stage during the plant life cycle in which a unicellular zygote develops into a multicellular organism. Co-ordinated gene expression is thus necessary for proper embryo development. Polycomb and Trithorax group genes are members of evolutionarily conserved machinery that maintains the correct expression patterns of key developmental regulators by repressing and activating gene transcription. TRAUCO (TRO), a gene homologous to the Trithorax group of genes that can functionally complement a BRE2P yeast mutant, has been identified in Arabidopsis thaliana. It is demonstrated that TRO is a nuclear gene product expressed during embryogenesis, and loss of TRO function leads to impaired early embryo development. Embryos that arrested at the globular stage in the tro-1 mutant allele were fully rescued by a TRO expression clone, a demonstration that the tro-1 mutation is a true loss-of-function in TRO. Our data have established that TRO is the first trithorax-group gene homologue in plants that is required for early embryogenesis.

In Arabidopsis thaliana, the three MADS box genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1), and SHP2 redundantly regulate ovule development. Protein interaction studies have shown that a multimeric complex composed of the ovule identity proteins together with the SEPALLATA MADS domain proteins is necessary to determine ovule identity. Despite the extensive knowledge that has become available about these MADS domain transcription factors, little is known regarding the genes that they regulate. Here, we show that STK, SHP1, and SHP2 redundantly regulate VERDANDI (VDD), a putative transcription factor that belongs to the plant-specific B3 superfamily. The vdd mutant shows defects during the fertilization process resulting in semisterility. Analysis of the vdd mutant female gametophytes indicates that antipodal and synergid cell identity and/or differentiation are affected. Our results provide insights into the pathways regulated by the ovule identity factors and the role of the downstream target gene VDD in female gametophyte development.

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.

SummaryThe development of multicellular organisms is controlled by differential gene expression whereby cells adopt distinct fates. A spatially resolved view of gene expression allows the elucidation of transcriptional networks that are linked to cellular identity and function. The haploid female gametophyte of flowering plants is a highly reduced organism: at maturity, it often consists of as few as three cell types derived from a common precursor [1, 2]. However, because of its inaccessibility and small size, we know little about the molecular basis of cell specification and differentiation in the female gametophyte. Here we report expression profiles of all cell types in the mature Arabidopsis female gametophyte. Differentially expressed posttranscriptional regulatory modules and metabolic pathways characterize the distinct cell types. Several transcription factor families are overrepresented in the female gametophyte in comparison to other plant tissues, e.g., type I MADS domain, RWP-RK, and reproductive meristem transcription factors. PAZ/Piwi-domain encoding genes are upregulated in the egg, indicating a role of epigenetic regulation through small RNA pathways—a feature paralleled in the germline of animals [3]. A comparison of human and Arabidopsis egg cells for enrichment of functional groups identified several similarities that may represent a consequence of coevolution or ancestral gametic features.

We have isolated seven Drosophila genes by means of low-stringency hybridization to a DNA probe containing the coding sequence for the protein domain shared by the rodent hepatocyte-enriched nuclear transcription factor HNF3A (alpha) and the product of the Drosophila region-specific homeotic gene fork head (fkh). The previously unreported genes encode a 110-amino acid conserved sequence, which we call the fork head (fkh) domain. Two of these fkh-domain-encoding genes ("FD genes") map to the sloppy paired locus (slp), which exerts segmentation gene function. The expression patterns of the other FD genes suggest that their protein products are likely to be involved in gut formation, mesoderm specification, and some specific aspects of neural development. The FD gene products presumably represent a family of transcription factors that, like the previously identified DNA-binding proteins, contribute to early developmental decisions in cell fates during embryogenesis.

Pollen, the male gametophyte of flowering plants, represents an ideal biological system to study developmental processes, such as cell polarity, tip growth, and morphogenesis. Upon hydration, the metabolically quiescent pollen rapidly switches to an active state, exhibiting extremely fast growth. This rapid switch requires relevant proteins to be stored in the mature pollen, where they have to retain functionality in a desiccated environment. Using a shotgun proteomics approach, we unambiguously identified ∼3500 proteins in Arabidopsis pollen, including 537 proteins that were not identified in genetic or transcriptomic studies. To generate this comprehensive reference data set, which extends the previously reported pollen proteome by a factor of 13, we developed a novel deterministic peptide classification scheme for protein inference. This generally applicable approach considers the gene model–protein sequence–protein accession relationships. It allowed us to classify and eliminate ambiguities inherently associated with any shotgun proteomics data set, to report a conservative list of protein identifications, and to seamlessly integrate data from previous transcriptomics studies. Manual validation of proteins unambiguously identified by a single, information-rich peptide enabled us to significantly reduce the false discovery rate, while keeping valuable identifications of shorter and lower abundant proteins. Bioinformatic analyses revealed a higher stability of pollen proteins compared to those of other tissues and implied a protein family of previously unknown function in vesicle trafficking. Interestingly, the pollen proteome is most similar to that of seeds, indicating physiological similarities between these developmentally distinct tissues.

Summary The screening of enhancer detector lines in Arabidopsis thaliana has identified genes that are specifically expressed in the sporophytic tissue of the ovule. One such gene is the MADS-domain transcription factor AGAMOUS-LIKE6 (AGL6), which is expressed asymmetrically in the endothelial layer of the ovule, adjacent to the developing haploid female gametophyte. Transcription of AGL6 is regulated at multiple stages of development by enhancer and silencer elements located in both the upstream regulatory region and the large first intron. These include a bipartite enhancer, which requires elements in both the upstream regulatory region and the first intron, active in the endothelium. Transcription of the AGL13 locus, which encodes the other member of the AGL6 subfamily in Arabidopsis, is also regulated by elements located in the upstream regulatory region and in the first intron. There is, however, no overlapping expression of AGL6 and AGL13 except in the chalaza of the developing ovule, as was shown using a dual gene reporter system. Phylogenetic shadowing of the first intron of AGL6 and AGL13 homologs from other Brassicaceae identified four regions of conservation that probably contain the binding sites of transcriptional regulators, three of which are conserved outside Brassicaceae. Further phylogenetic analysis using the protein-encoding domains of AGL6 and AGL13 revealed that the MADS DNA-binding domain shows considerable divergence. Together, these results suggest that AGL6 and AGL13 show signs of subfunctionalization, with divergent expression patterns, regulatory sequences and possibly functions. Journal compilation � 2009 Blackwell Publishing Ltd.

The precise delivery of male to female gametes during reproduction in eukaryotes requires complex signal exchanges and a flawless communication between male and female tissues. In angiosperms, molecular mechanisms have recently been revealed that are crucial for the dialog between male (pollen tube) and female gametophytes required for successful sperm delivery. When pollen tubes reach the female gametophyte, they arrest growth, burst and discharge their sperm cells. These processes are under the control of the female gametophyte via the receptor-like serine-threonine kinase (RLK) FERONIA (FER). However, the male signaling components that control the sperm delivery remain elusive. Here, we show that ANXUR1 and ANXUR2 (ANX1, ANX2), which encode the closest homologs of the FER-RLK in Arabidopsis, are preferentially expressed in pollen. Moreover, ANX1-YFP and ANX2-YFP fusion proteins display polar localization to the plasma membrane at the tip of the pollen tube. Finally, genetic analyses demonstrate that ANX1 and ANX2 function redundantly to control the timing of pollen tube discharge as anx1 anx2 double-mutant pollen tubes cease their growth and burst in vitro and fail to reach the female gametophytes in vivo. We propose that ANX-RLKs constitutively inhibit pollen tube rupture and sperm discharge at the tip of growing pollen tubes to sustain their growth within maternal tissues until they reach the female gametophytes. Upon arrival, the female FER-dependent signaling cascade is activated to mediate pollen tube reception and fertilization, while male ANX-dependent signaling is deactivated, enabling the pollen tube to rupture and deliver its sperm cells to effect fertilization.

We have assembled a proteome map for Arabidopsis thaliana from high-density, organ-specific proteome catalogs that we generated for different organs, developmental stages, and undifferentiated cultured cells. We matched 86,456 unique peptides to 13,029 proteins and provide expression evidence for 57 gene models that are not represented in the TAIR7 protein database. Analysis of the proteome identified organ-specific biomarkers and allowed us to compile an organ-specific set of proteotypic peptides for 4105 proteins to facilitate targeted quantitative proteomics surveys. Quantitative information for the identified proteins was used to establish correlations between transcript and protein accumulation in different plant organs. The Arabidopsis proteome map provides information about genome activity and proteome assembly and is available as a resource for plant systems biology.

In the animal kingdom, maternal control of early development is a common feature. The onset of zygotic control over early development, defined as the maternal to zygotic transition (MZT), follows fertilization with a delay of a variable number of cell divisions, depending on the species. The MZT has been well defined in animals, but investigations remain in their infancy in plants. Recent evidence suggests, however, that in plants as in animals, the MZT also occurs several division cycles after fertilization. The likely convergent evolution of the MZT in the animal and plant kingdoms is fascinating and raises major questions regarding its biological significance, particularly with regard to its importance in genome reprogramming and the acquisition of totipotency by the embryo.

MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.

The sperm cell of flowering plants cannot migrate unaided and must be transported by the pollen tube cell of the male gametophyte to achieve successful fertilization. Long-distance pollen tube guidance is controlled by the seven-celled female gametophyte, the embryo sac. Previous reports showed that the synergid cell of the embryo sac is essential for pollen tube guidance. Here, we report the identification of a central cell guidance (ccg) mutant, which is defective in micropylar pollen tube guidance. CCG encodes a nuclear protein with an N-terminal conserved zinc β-ribbon domain that is functionally interchangeable with that of TFIIB in yeast. This suggests that CCG might act as a transcription regulator for pollen tube guidance. CCG is expressed in the central cell of the female gametophyte. Expression of CCG in the central cell alone is sufficient to restore the normal pollen tube guidance phenotype, demonstrating that the central cell plays a critical role in pollen tube guidance.

Genetic subtraction and expression profiling of wild-type Arabidopsis and a sporophytic mutant lacking an embryo sac identified 1,260 genes expressed in the embryo sac; a total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac., Background The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis. Results We identified 1,260 genes as embryo sac expressed by analyzing both our dataset and a recently reported dataset, obtained by a similar approach, using three statistical procedures. Spatial expression of nine genes (for instance a central cell expressed trithorax-like gene, an egg cell expressed gene encoding a kinase, and a synergid expressed gene encoding a permease) validated our approach. We analyzed mutants in five of the newly identified genes that exhibited developmental anomalies during reproductive development. A total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac, at levels that were twofold higher than in the wild type. Conclusion Identification of embryo sac expressed genes establishes a basis for the functional dissection of embryo sac development and function. Sporophytic gain of expression in mutants lacking an embryo sac suggests that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac.

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3' to 5' trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations.

Our understanding of nearly all aspects of plant growth and development have benefited from the application of genetic analyses. Early anatomical and physiological studies provided evidence for processes that allow the plant to respond to internal and external stimuli, but genetics has provided the tools to revolutionize our understanding of the underlying mechanisms. The development of new tools in molecular genetics, the adoption of plant model organisms and the creation of databases for comparative gene analyses, have all contributed to the rapid progress in our understanding of plant developmental biology in recent years. Keywords: Arabidopsis thaliana; dominance; epistasis; genetic screen; mutation; positional cloning

Seed development starts at double fertilization when two sperms fuse with a female gamete, the egg and central cell, giving rise to the embryo and endosperm, respectively. Uniting two parental genomes into one, unique, zygotic genome is certainly the first event requiring large-scale chromatin modifications and remodeling. Although little is known about the molecular mechanisms, recent progress was made allowing live imaging of parental chromatin dynamics at fertilization. Further growth and patterning processes will shape the future plant seedling and its surrounding nurse tissue. Studies over the last decade identified key chromatin modifiers involved in these processes. However, the dynamics of these modifications mediated, in particular, by the Polycomb group complexes only start to be understood. The precise molecular mechanisms altering chromatin state in relation to early seed development remains difficult to address because of the relative inaccessibility of the fertilization products. Nonetheless, with the emergence of in vivo imaging techniques, laser-capture dissection, and genome-wide chromatin modification profiling, the future promises new, exciting discoveries.

The endosperm is a seed tissue unique to flowering plants. Due to its central role in nourishing and protecting the embryo, endosperm development is subject to parental conflicts and adaptive processes, which led to the evolution of parent-of-origin–dependent gene regulation. The role of higher-order chromatin organization in regulating the endosperm genome was long ignored due to technical hindrance. We developed a combination of approaches to analyze nuclear structure and chromatin organization in Arabidopsis thaliana endosperm. Endosperm nuclei showed a less condensed chromatin than other types of nuclei and a peculiar heterochromatin organization, with smaller chromocenters and additional heterochromatic foci interspersed in euchromatin. This is accompanied by a redistribution of the heterochromatin mark H3K9me1 from chromocenters toward euchromatin and interspersed heterochromatin. Thus, endosperm nuclei have a specific nuclear architecture and organization, which we interpret as a relaxed chromocenter-loop model. The analysis of endosperm with altered parental genome dosage indicated that the additional heterochromatin may be predominantly of maternal origin, suggesting differential regulation of maternal and paternal genomes, possibly linked to genome dosage regulation.

In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis) mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions., Author Summary The selection and specification of the egg cell determine the number of eggs produced by an animal or plant, which in turn dictates how many offspring that organism can produce. In most higher plants, the egg cell forms in a specialized structure consisting of four different cell types. Two cells, the egg cell and the central cell, are fertilized by sperm cells and develop into the embryo proper and the nutritive tissue (endosperm), respectively. These two gametic cells are flanked by accessory cells; but why do some cells become gametic while others differentiate into accessory cells? To answer this question, we looked for mutants in which this process is disturbed. In the lachesis mutant, accessory cells become extra egg cells. Interestingly, it seems that the misspecification of these accessory cells results from defects in the gametic cells. This suggests that accessory cells monitor the state of the gametic cells to act as a backup if required, ensuring the formation of the key reproductive cells., In plant egg cells, gametophytes differentiate into both gametic and accessory cells; here the authors characterize a mutant that turns accessory cells into gametic cells.

Pollen tube (PT) reception in flowering plants describes the crosstalk between the male and female gametophytes upon PT arrival at the synergid cells of the ovule. It leads to PT growth arrest, rupture, and sperm cell release, and is thus essential to ensure double fertilization. Here, we describe TURAN (TUN) and EVAN (EVN), two novel members of the PT reception pathway that is mediated by the FERONIA (FER) receptor-like kinase (RLK). Like fer, mutations in these two genes lead to PT overgrowth inside the female gametophyte (FG) without PT rupture. Mapping by next-generation sequencing, cytological analysis of reporter genes, and biochemical assays of glycoproteins in RNAi knockdown mutants revealed both genes to be involved in protein N-glycosylation in the endoplasmic reticulum (ER). TUN encodes a uridine diphosphate (UDP)-glycosyltransferase superfamily protein and EVN a dolichol kinase. In addition to their common role during PT reception in the synergids, both genes have distinct functions in the pollen: whereas EVN is essential for pollen development, TUN is required for PT growth and integrity by affecting the stability of the pollen-specific FER homologs ANXUR1 (ANX1) and ANX2. ANX1- and ANX2-YFP reporters are not expressed in tun pollen grains, but ANX1-YFP is degraded via the ER-associated degradation (ERAD) pathway, likely underlying the anx1/2-like premature PT rupture phenotype of tun mutants. Thus, as in animal sperm–egg interactions, protein glycosylation is essential for the interaction between the female and male gametophytes during PT reception to ensure fertilization and successful reproduction., Protein glycosylation is essential for gametophyte interactions between the male pollen tube and the female ovule in plants, reminiscent of gamete interactions during fertilization in mammals., Author Summary In flowering plants, gametes are produced by the haploid, multicellular male (pollen), and female (embryo sac) gametophytes, which develop within the reproductive organs of the flower. Successful fertilization depends on delivery of the sperm cells to the embryo sac, which is embedded in the ovule, by the pollen tube. Upon arrival of the pollen tube at the opening of the ovule, crosstalk between male and female gametophytes, known as pollen tube reception, ensues; the pollen tube slows or stops its growth, then resumes rapid growth, and finally bursts to release the sperm cells and effect double fertilization. Although several members of the pollen tube reception pathway, including the receptor-like kinase FERONIA, have been identified, the molecular mechanisms underlying this communication process remain unclear. Here, we show that protein N-glycosylation is required for normal pollen tube reception. A mutant screen identified two genes, TURAN and EVAN, which are involved in protein N-glycosylation in the endoplasmic reticulum. Both genes act in the FERONIA-mediated pollen tube reception pathway, which is impaired in these mutants. Thus, in plants, a “dual recognition system,” involving interactions between both protein and glycosyl residues on the surface of male and female gametophytes, appears to be required for successful pollen tube reception, conceptually similar to sperm–egg interactions in mammals, for which N-glycosylation of cell surface proteins also plays an important role.

Imprinted gene expression refers to differential transcription of alleles depending on their parental origin. To date, most examples of imprinted gene expression in plants occur in the triploid endosperm tissue. The Arabidopsis gene MEDEA displays an imprinted pattern of gene expression and has homology to the Drosophila Polycomb group (PcG) protein Enhancer-of-zeste (E(z)). We have tested the allele-specific expression patterns of the three maize E(z)-like genes Mez1, Mez2 and Mez3. The expression of Mez2 and Mez3 is not imprinted, with a bi-allelic pattern of transcription for both genes in both the endosperm and embryonic tissue. In contrast, Mez1 displays a bi-allelic expression pattern in the embryonic tissue, and a mono-allelic expression pattern in the developing endosperm tissue. We demonstrate that mono-allelic expression of the maternal Mez1 allele occurs throughout endosperm development. We have identified a 556 bp differentially methylated region (DMR) located approximately 700 bp 5' of the Mez1 transcription start site. This region is heavily methylated at CpG and CpNpG nucleotides on the non-expressed paternal allele but has low levels of methylation on the expressed maternal allele. Molecular evolutionary analysis indicates that conserved domains of all three Mez genes are under purifying selection. The common imprinted expression of Mez1 and MEDEA, in concert with their likely evolutionary origins, suggests that there may be a requirement for imprinting of at least one E(z)-like gene in angiosperms.

The genome of the Thale Cress, Arabidopsis thaliana , was the first plant genome to be fully sequenced, providing a tool for the rapid characterization of biological processes. This versatile model organism has had a major impact on our understanding of the physiology, growth, and development of flowering plants, and their response to environmental stress. An in-depth understanding of model organisms, coupled with knowledge of crop systems, will provide rapid advancements in technology, including genetic engineering, that are required to overcome future predicted global climate changes and short falls in food production. Keywords: Abaxial; Accession; Acetylation; Adaxial; ADP-ribosylation; Angiosperms; Apomict; Bacterial Artificial Chromosomes (BACs); Centromere; Cleaved Amplified Polymorphic Sequences (CAPS); Colinearity; Dicots; Dioecious Plants; Diploid; DNA Polymorphism; Ectopic Expression; Epigenetics; Euchromatin; Exon; Expressed Sequenced Tags; Forward Genetics; Gametophyte; Genetic Map; Haploid; Hermaphroditic Plants; Heterochromatin; Hexaploid; Histone; Homeotic Genes; Intron; Linkage Groups; Locus; Methylation; Monocots; Monoecious Plants; Morphological; Nucleosome; Orthologous Genes; ParalogousGenes; Pericentromeric; Phosphorylation; Phylogenetic Relationship; Physical Map; Polyploid; Rapid Amplified Polymorphic DNA; Recombination; Retroelements; Reverse Genetics; Satellite Repeat; Simple Sequence Length Polymorphisms (SSLP); Sumoylation; Synteny; Telomeres; Tiling Path; Transformation; Transposons; Ubiquitination; Vernalization; Yeast Artificial Chromosomes (YACs)

MADS-domain transcription factors are essential for proper flower and seed development in angiosperms and their role in determination of floral organ identity can be described by the 'ABC model' of flower development. Recently, close relatives of the B-type genes were identified by phylogenetic studies, which are referred to as Bsister (Bs) genes. Here, we report the isolation and characterization of a MADS-box Bs member from petunia, designated FBP24. An fbp24 knock-down line appeared to closely resemble the Arabidopsis Bs mutant abs and a detailed and comparative analysis led to the conclusion that both FBP24 and ABS are necessary to determine the identity of the endothelial layer within the ovule. Protein interaction studies revealed the formation of higher-order complexes between Bs-C-E and Bs-D-E type MADS-box proteins, suggesting involvement of these specific complexes in determination of endothelium identity. However, although there are many similarities between the two genes and their products and functions, interestingly FBP24 cannot replace ABS in Arabidopsis. The results presented here demonstrate the importance of the comparative analysis of key regulatory genes in various model systems to fully understand all aspects of plant development. � 2006 The Authors.

In flowering plants, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes that regulate anther cell differentiation and function. Here, we report the analysis of a new Arabidopsis male sterile mutant, dysfunctional tapetum1 (dyt1). The dyt1 mutant exhibits abnormal anther morphology beginning at anther stage 4, with tapetal cells that have excess and/or enlarged vacuoles and lack the densely stained cytoplasm typical of normal tapetal cells. The mutant meiocytes are able to complete meiosis I,but they do not have a thick callose wall; they often fail to complete meiotic cytokinesis and eventually collapse. DYT1 encodes a putative bHLH transcription factor and is strongly expressed in the tapetum from late anther stage 5 to early stage 6, and at a lower level in meiocytes. In addition, the level of DYT1 mRNA is reduced in the sporocyteless/nozzle(spl/nzz) and excess microsporocytes1/extra sporogenous cell(ems1/exs) mutants; together with the mutant phenotypes, this suggests that DYT1 acts downstream of SPL/NZZ and EMS1/EXS. RT-PCR results showed that the expression levels of many tapetum-preferential genes are reduced significantly in the dyt1mutant, indicating that DYT1 is important for the expression of tapetum genes. Our results support the hypothesis that DYT1 is a crucial component of a genetic network that controls anther development and function.

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.

The 26S proteasome plays a central role in the degradation of regulatory proteins involved in a variety of developmental processes. It consists of two multisubunit protein complexes: the proteolytic core protease and the regulatory particle (RP). The function of most RP subunits is poorly understood. Here, we describe mutants in the Arabidopsis thaliana RPN1 subunit, which is encoded by two paralogous genes, RPN1a and RPN1b. Disruption of RPN1a caused embryo lethality, while RPN1b mutants showed no obvious abnormal phenotype. Embryos homozygous for rpn1a arrested at the globular stage with defects in the formation of the embryonic root, the protoderm, and procambium. Cyclin B1 protein was not degraded in these embryos, consistent with cell division defects. Double mutant plants (rpn1a/RPN1a rpn1b/rpn1b) produced embryos with a phenotype indistinguishable from that of the rpn1a single mutant. Thus, despite their largely overlapping expression patterns in flowers and developing seeds, the two isoforms do not share redundant functions during gametogenesis and embryogenesis. However, complementation of the rpn1a mutation with the coding region of RPN1b expressed under the control of the RPN1a promoter indicates that the two RPN1 isoforms are functionally equivalent. Overall, our data indicate that RPN1 activity is essential during embryogenesis, where it might participate in the destruction of a specific set of protein substrates.

Genomic imprinting refers to an epigenetic phenomenon where the activity of an allele depends on its parental origin. Imprinting at individual genes has only been described in mammals and seed plants. We will discuss the role imprinted genes play in seed development and compare the situation in plants with that in mammals. Interestingly, many imprinted genes appear to control cell proliferation and growth in both groups of organisms although imprinting in plants may also be involved in the cellular differentiation of the two pairs of gametes involved in double fertilization. DNA methylation plays some role in the control of parent-of-origin-specific expression in both mammals and plants. Thus, although imprinting evolved independently in mammals and plants, there are striking similarities at the phenotypic and possibly also mechanistic level.

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.

Genomic imprinting refers to an epigenetic phenomenon where the activity of an allele depends on its parental origin. Imprinting at individual genes has only been described in mammals and seed plants. We will discuss the role imprinted genes play in seed development and compare the situation in plants with that in mammals. Interestingly, many imprinted genes appear to control cell proliferation and growth in both groups of organisms although imprinting in plants may also be involved in the cellular differentiation of the two pairs of gametes involved in double fertilization. DNA methylation plays some role in the control of parent-of-origin-specific expression in both mammals and plants. Thus, although imprinting evolved independently in mammals and plants, there are striking similarities at the phenotypic and possibly also mechanistic level.

To identify genes with essential roles in male gametophytic development, including postpollination (progamic) events, we have undertaken a genetic screen based on segregation ratio distortion of a transposon-borne kanamycin-resistance marker. In a population of 3359 Arabidopsis Ds transposon insertion lines, we identified 20 mutants with stably reduced segregation ratios arising from reduced gametophytic transmission. All 20 mutants showed strict cosegregation of Ds and the reduced gametophytic transmission phenotype. Among these, 10 mutants affected both male and female transmission and 10 mutants showed male-specific transmission defects. Four male and female (ungud) mutants and 1 male-specific mutant showed cellular defects in microspores and/or in developing pollen. The 6 remaining ungud mutants and 9 male-specific (seth) mutants affected pollen functions during progamic development. In vitro and in vivo analyses are reported for 5 seth mutants. seth6 completely blocked pollen germination, while seth7 strongly reduced pollen germination efficiency and tube growth. In contrast, seth8, seth9, or seth10 pollen showed reduced competitive ability that was linked to slower rates of pollen tube growth. Gene sequences disrupted in seth insertions suggest essential functions for putative SETH proteins in diverse processes including protein anchoring, cell wall biosynthesis, signaling, and metabolism.

Apomixis is the process of asexual reproduction through seed, in the absence of meiosis and fertilization, generating clonal progeny of maternal origin. Major benefits to agriculture could result from harnessing apomixis in crop plants. Although >400 apomictic plant species are known, apomixis is rare among crop plants, and the transfer of apomixis to crop varieties by conventional breeding has been largely unsuccessful. Because apomictic and sexual pathways are closely related, de novo engineering of apomixis might be achieved in sexually reproducing crops. Early consideration of issues relating to biosafety and intellectual property (IP) management can facilitate the acceptance and deployment of apomixis technology in agriculture.

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thalianainvolves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA(MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM(FIE). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia. We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.

Times Cited: 38 Cited Reference Count: 67; International audience; The life cycles of sexually reproducing animals and flowering plants begin with male and female gametes and their fusion to form a zygote. Selection at this earliest stage is crucial for offspring quality and raises similar evolutionary issues, yet zoology and botany use dissimilar approaches. There are striking parallels in the role of prezygotic competition for sexual selection on males, cryptic female choice, sexual conflict, and against selfish genetic elements and genetic incompatibility. In both groups, understanding the evolution of sex-specific and reproductive traits will require an appreciation of the effects of prezygotic competition on fitness.

Important aspects of plant development are under epigenetic control, that is, under the control of heritable changes in gene expression that are not associated with alterations in DNA sequence. It is becoming clear that RNA molecules play a key role in epigenetic gene regulation by providing sequence specificity for the targeting of developmentally important genes. RNA-based control of gene expression can be exerted posttranscriptionally by interfering with transcript stability or translation. Moreover, RNA molecules also appear to direct developmentally relevant gene regulation at the transcriptional level by modifying chromatin structure and/or DNA methylation.

The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the β-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.

The term apomixis encompasses a suite of processes whereby seeds form asexually in plants. In contrast to sexual reproduction, seedlings arising from apomixis retain the genotype of the maternal parent. The transfer of apomixis and its effective utilization in crop plants (where it is largely absent) has major advantages in agriculture. The hallmark components of apomixis include female gamete formation without meiosis (apomeiosis), fertilization-independent embryo development (parthenogenesis), and developmental adaptations to ensure functional endosperm formation. Understanding the molecular mechanisms underlying apomixis, a developmentally fascinating phenomenon in plants, is critical for the successful induction and utilization of apomixis in crop plants. This review draws together knowledge gained from analyzing ovule, embryo, and endosperm development in sexual and apomictic plants. It consolidates the view that apomixis and sexuality are closely interrelated developmental pathways where apomixis can be viewed as a deregulation of the sexual process in both time and space.

Reproduction in angiosperms depends on communication processes of the male gametophyte (pollen) with the female floral organs (pistil, transmitting tissue) and the female gametophyte (embryo sac). Pollen-pistil interactions control pollen hydration, germination and growth through the stylar tissue. The female gametophyte is involved in guiding the growing pollen tube towards the micropyle and embryo sac. One of the two synergids flanking the egg cell starts to degenerate and becomes receptive for pollen tube entry. Pollen tube growth arrests and the tip of the pollen tube ruptures to release the sperm cells. Failures in the mutual interaction between the synergid and the pollen tube necessarily impair fertility. But the control of pollen tube reception is not understood. We isolated a semisterile, female gametophytic mutant from Arabidopsis thaliana, named feronia after the Etruscan goddess of fertility, which impairs this process. In the feroniamutant, embryo sac development and pollen tube guidance were unaffected in all ovules, although one half of the ovules bore mutant female gametophytes. However, when the pollen tube entered the receptive synergid of a feronia mutant female gametophyte, it continued to grow, failed to rupture and release the sperm cells, and invaded the embryo sac. Thus, the feronia mutation disrupts the interaction between the male and female gametophyte required to elicit these processes. Frequently, mutant embryo sacs received supernumerary pollen tubes. We analysed feronia with synergid-specific GUS marker lines, which demonstrated that the specification and differentiation of the synergids was normal. However, GUS expression in mutant gametophytes persisted after pollen tube entry, in contrast to wild-type embryo sacs where it rapidly decreased. Apparently, the failure in pollen tube reception results in the continued expression of synergid-specific genes, probably leading to an extended expression of a potential pollen tube attractant.

Polycomb group (PcG) proteins play essential roles in animal and plant life cycles by controlling the expression of important developmental regulators. These structurally heterogeneous proteins form multimeric protein complexes that control higher order chromatin structure and, thereby, the expression state of their target genes. Once established, PcG proteins maintain silent gene expression states over many cell divisions providing a molecular basis for a cellular 'memory.' PcG proteins are best known for their role in the control of homeotic genes in Drosophila and mammals. In addition, they play important roles in the control of cell proliferation in vertebrate and invertebrate systems. Recent studies in plants have shown that PcG proteins regulate diverse developmental processes and, as in animals, they affect both homeotic gene expression and cell proliferation. Thus, the function of PcG proteins has been widely conserved between the plant and animal kingdoms.

The development of apomixis technology in crop plants is a desirable goal. Apomixis is the asexual reproduction through seeds, which occurs in over 400 flowering plants (Nogler, 1984). The introduction of clonal reproduction to crop plants will allow the indefinite propagation of any desirable genotype (including that of heterozygous F1 hybrids) and will completely transform current breeding and seed production strategies. Developmental aspects of apomixis (Koltunow, 1993; Grossniklaus, 2001; Spillane et al., 2001), its genetic control (Savidan, 2000; Grossniklaus et al., 2001a; Grimanelli et al., 2001), and its potential use in agriculture (Koltunow et al., 1995; Hanna et al., 1998; Jefferson and Bicknell, 1996; Thoenissen, 2001) have been extensively reviewed. Here, we provide a short summary of developmental and genetic aspects and report on our program using sexual model systems to identify genes and promoters relevant to the engineering of apomixis.