Jacqueline Gheyselinck

Arabidopsis MSI1 is a component of the MEA/FIE Polycomb group complex and required for seed development.

Seed development in angiosperms initiates after double fertilization, leading to the formation of a diploid embryo and a triploid endosperm. The active repression of precocious initiation of certain aspects of seed development in the absence of fertilization requires the Polycomb group proteins MEDEA (MEA), FERTILIZATION‐INDEPENDENT ENDOSPERM (FIE) and FERTILIZATION‐INDEPENDENT SEED2. Here we show that the Arabidopsis WD‐40 domain protein MSI1 is present together with MEA and FIE in a 600 kDa complex and interacts directly with FIE. Mutant plants heterozygous for msi1 show a seed abortion ratio of 50% with seeds aborting when the mutant allele is maternally inherited, irrespective of a paternal wild‐type or mutant MSI1 allele. Further more, msi1 mutant gametophytes initiate endosperm development in the absence of fertilization at a high penetrance. After pollination, only the egg cell becomes fertilized, the central cell starts dividing prior to fertilization, resulting in the formation of seeds containing embryos surrounded by diploid endosperm. Our results establish that MSI1 has an essential function in the correct initiation and progression of seed development.

Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness

In plants, members of microRNA (miRNA) families are often predicted to target the same or overlapping sets of genes. It has thus been hypothesized that these miRNAs may act in a functionally redundant manner. This hypothesis is tested here by studying the effects of elimination of all three members of the MIR164 family from Arabidopsis. It was found that a loss of miR164 activity leads to a severe disruption of shoot development, in contrast to the effect of mutation in any single MIR164 gene. This indicates that these miRNAs are indeed functionally redundant. Differences in the expression patterns of the individual MIR164 genes imply, however, that redundancy among them is not complete, and that these miRNAs show functional specialization. Furthermore, the results of molecular and genetic analyses of miR164-mediated target regulation indicate that miR164 miRNAs function to control the transcript levels, as well as the expression patterns, of their targets, suggesting that they might contribute to developmental robustness. For two of the miR164 targets, namely CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, we provide evidence for their involvement in the regulation of growth and show that their derepression in miR164 loss-of-function mutants is likely to account for most of the mutant phenotype.

Arabidopsis Female Gametophyte Gene Expression Map Reveals Similarities between Plant and Animal Gametes

The 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.

Egg Cell–Secreted EC1 Triggers Sperm Cell Activation During Double Fertilization

Double Delivery During Plant Fertilization Double fertilization is a defining feature of flowering plants and involves two nonmotile male gametes (sperm cells) and two female gametes (egg cell and central cell). Both fertilization events are necessary for reproductive success. It is not clear how flowering plants ensure the reliable and on-time fusion of the two pairs of gametes, while preventing polyspermy. Sprunck et al. (p. 1093; see the Perspective by Snell) now show that gamete interactions in Arabidopsis depend on small cysteine-rich EGG CELL 1 (EC1) proteins that accumulate in storage vesicles of the egg cell and that are released during sperm-egg interaction. EC1 peptides trigger the delivery of a fusogen to the sperm cell surface. An intercellular link connects the two sperm cells throughout the gamete fusion process and could play a role in preventing the spontaneous fusion of activated sperm cells. The cysteine-rich proteins of Arabidopsis egg and central cells enable fusion with just one sperm each. Double fertilization is the defining characteristic of flowering plants. However, the molecular mechanisms regulating the fusion of one sperm with the egg and the second sperm with the central cell are largely unknown. We show that gamete interactions in Arabidopsis depend on small cysteine-rich EC1 (EGG CELL 1) proteins accumulating in storage vesicles of the egg cell. Upon sperm arrival, EC1-containing vesicles are exocytosed. The sperm endomembrane system responds to exogenously applied EC1 peptides by redistributing the potential gamete fusogen HAP2/GCS1 (HAPLESS 2/GENERATIVE CELL SPECIFIC 1) to the cell surface. Furthermore, fertilization studies with ec1 quintuple mutants show that successful male-female gamete interactions are necessary to prevent multiple–sperm cell delivery. Our findings provide evidence that mutual gamete activation, regulated exocytosis, and sperm plasma membrane modifications govern flowering plant gamete interactions.

Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female gametophyte and the maternal sporophyte

BackgroundThe 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.ResultsWe 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.ConclusionIdentification 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 RPN1 Subunit of the 26S Proteasome in Arabidopsis Is Essential for Embryogenesis

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.

Members of the RKD transcription factor family induce an egg cell‐like gene expression program

In contrast to animals, the life cycle of higher plants alternates between a gamete-producing (gametophyte) and a spore-producing (sporophyte) generation. The female gametophyte of angiosperms consists of four distinct cell types, including two gametes, the egg and the central cell, which give rise to embryo and endosperm, respectively. Based on a combined subtractive hybridization and virtual subtraction approach in wheat (Triticum aestivum L.), we have isolated a class of transcription factors not found in animal genomes, the RKD (RWP-RK domain-containing) factors, which share a highly conserved RWP-RK domain. Single-cell RT-PCR revealed that the genes TaRKD1 and TaRKD2 are preferentially expressed in the egg cell of wheat. The Arabidopsis genome contains five RKD genes, at least two of them, AtRKD1 and AtRKD2, are preferentially expressed in the egg cell of Arabidopsis. Ectopic expression of the AtRKD1 and AtRKD2 genes induces cell proliferation and the expression of an egg cell marker. Analyses of RKD-induced proliferating cells exhibit a shift of gene expression towards an egg cell-like transcriptome. Promoters of selected RKD-induced genes were shown to be predominantly active in the egg cell and can be activated by RKD in a transient protoplast expression assay. The data show that egg cell-specific RKD factors control a transcriptional program, which is characteristic for plant egg cells.

Intronic regulatory elements determine the divergent expression patterns of AGAMOUS‐LIKE6 subfamily members in Arabidopsis

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.

Genetic Interaction of an Origin Recognition Complex Subunit and the Polycomb Group Gene MEDEA during Seed Development

The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.

Engineering of Apomixis in Crop Plants: What Can We Learn from Sexual Model Systems?

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.