Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development.

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.

Fluorescent in situ hybridization shows DIPLOSPOROUS located on one of the NOR chromosomes in apomictic dandelions (Taraxacum) in the absence of a large hemizygous chromosomal region.

Apomixis in dandelions (Taraxacum: Asteraceae) is encoded by two unlinked dominant loci and a third yet undefined genetic factor: diplosporous omission of meiosis (DIPLOSPOROUS, DIP), parthenogenetic embryo development (PARTHENOGENESIS, PAR), and autonomous endosperm formation, respectively. In this study, we determined the chromosomal position of the DIP locus in Taraxacum by using fluorescent in situ hybridization (FISH) with bacterial artificial chromosomes (BACs) that genetically map within 1.2-0.2 cM of DIP. The BACs showed dispersed fluorescent signals, except for S4-BAC 83 that displayed strong unique signals as well. Under stringent blocking of repeats by C0t-DNA fragments, only a few fluorescent foci restricted to defined chromosome regions remained, including one on the nucleolus organizer region (NOR) chromosomes that contains the 45S rDNAs. FISH with S4-BAC 83 alone and optimal blocking showed discrete foci in the middle of the long arm of one of the NOR chromosomes only in triploid and tetraploid diplosporous dandelions, while signals in sexual diploids were lacking. This agrees with the genetic model of a single dose, dominant DIP allele, absent in sexuals. The length of the DIP region is estimated to cover a region of 1-10 Mb. FISH in various accessions of Taraxacum and the apomictic sister species Chondrilla juncea, confirmed the chromosomal position of DIP within Taraxacum but not outside the genus. Our results endorse that, compared to other model apomictic species, expressing either diplospory or apospory, the genome of Taraxacum shows a more similar and less diverged chromosome structure at the DIP locus. The different levels of allele sequence divergence at apomeiosis loci may reflect different terms of asexual reproduction. The association of apomeiosis loci with repetitiveness, dispersed repeats, and retrotransposons commonly observed in apomictic species may imply a functional role of these shared features in apomictic reproduction, as is discussed.

Phylogenomic analysis provides insights into MADS-box and TCP gene diversification and floral development of the Asteraceae, supported by de novo genome and transcriptome sequences from dandelion (Taraxacum officinale).

The Asteraceae is the largest angiosperm family with more than 25,000 species. Individual studies have shown that MADS-box and TCP transcription factors are regulators of the development and symmetry of flowers, contributing to their iconic flower-head (capitulum) and floret. However, a systematic study of MADS-box and TCP genes across the Asteraceae is lacking. We performed a comparative analysis of genome sequences of 33 angiosperm species including our de novo assembly of diploid sexual dandelion (Taraxacum officinale) and 11 other Asteraceae to investigate the lineage-specific evolution of MADS-box and TCP genes in the Asteraceae. We compared the phylogenomic results of MADS-box and TCP genes with their expression in T. officinale floral tissues at different developmental stages to demonstrate the regulation of genes with Asteraceae-specific attributes. Here, we show that MADS-box MIKC c and TCP-CYCLOIDEA (CYC) genes have expanded in the Asteraceae. The phylogenomic analysis identified AGAMOUS-like (AG-like: SEEDSTICK [STK]-like), SEPALATA-like (SEP3-like), and TCP-PROLIFERATING CELL FACTOR (PCF)-like copies with lineage-specific genomic contexts in the Asteraceae, Cichorioideae, or dandelion. Different expression patterns of some of these gene copies suggest functional divergence. We also confirm the presence and revisit the evolutionary history of previously named "Asteraceae-Specific MADS-box genes (AS-MADS)." Specifically, we identify non-Asteraceae homologs, indicating a more ancient origin of this gene clade. Syntenic relationships support that AS-MADS is paralogous to FLOWERING LOCUS C (FLC) as demonstrated by the shared ancient duplication of FLC and SEP3.

A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce.

Apomixis, the clonal formation of seeds, is a rare yet widely distributed trait in flowering plants. We have isolated the PARTHENOGENESIS (PAR) gene from apomictic dandelion that triggers embryo development in unfertilized egg cells. PAR encodes a K2-2 zinc finger, EAR-domain protein. Unlike the recessive sexual alleles, the dominant PAR allele is expressed in egg cells and has a miniature inverted-repeat transposable element (MITE) transposon insertion in the promoter. The MITE-containing promoter can invoke a homologous gene from sexual lettuce to complement dandelion LOSS OF PARTHENOGENESIS mutants. A similar MITE is also present in the promoter of the PAR gene in apomictic forms of hawkweed, suggesting a case of parallel evolution. Heterologous expression of dandelion PAR in lettuce egg cells induced haploid embryo-like structures in the absence of fertilization. Sexual PAR alleles are expressed in pollen, suggesting that the gene product releases a block on embryogenesis after fertilization in sexual species while in apomictic species PAR expression triggers embryogenesis in the absence of fertilization.

Sepal Identity of the Pappus and Floral Organ Development in the Common Dandelion (Taraxacum officinale; Asteraceae).

The dry one-seeded fruits (cypselae) of the Asteraceae are often crowned with a pappus, an appendage of hairs or scales that assists in dispersal. It is generally assumed, but little investigated, that the pappus represents the outer floral whorl where the sepals are usually located. We analysed pappus-sepal homology in dandelions using micromorphological and floral gene expression analyses. We show that the pappus initiates from a ring primordium at the base of the corolla, heterochronic to the petals. Pappus parts form from this ring, with those in the alternipetalaous position usually being ahead in growth, referring to sepal identity. Tof-APETALLA1 expression increased during floret development and was highest in mature pappus. Tof-PISTILLATA expression was high and confined to the floral tissues containing the petals and stamens, consistent with expectations for sepals. Apart from the pappus, the dispersal structure of dandelion consists of the upper part of the fruit, the beak, which originates from the inner floral whorl. Thus, our results support the homology of the pappus with the sepals, but show that it is highly derived. Together with our floral stage definitions and verified qPCR reference genes, they provide a basis for evolution and development studies in dandelions and possibly other Asteraceae.

Participation of Multifunctional RNA in Replication, Recombination and Regulation of Endogenous Plant Pararetroviruses (EPRVs).

Pararetroviruses, taxon Caulimoviridae, are typical of retroelements with reverse transcriptase and share a common origin with retroviruses and LTR retrotransposons, presumably dating back 1.6 billion years and illustrating the transition from an RNA to a DNA world. After transcription of the viral genome in the host nucleus, viral DNA synthesis occurs in the cytoplasm on the generated terminally redundant RNA including inter- and intra-molecule recombination steps rather than relying on nuclear DNA replication. RNA recombination events between an ancestral genomic retroelement with exogenous RNA viruses were seminal in pararetrovirus evolution resulting in horizontal transmission and episomal replication. Instead of active integration, pararetroviruses use the host DNA repair machinery to prevail in genomes of angiosperms, gymnosperms and ferns. Pararetrovirus integration - leading to Endogenous ParaRetroViruses, EPRVs - by illegitimate recombination can happen if their sequences instead of homologous host genomic sequences on the sister chromatid (during mitosis) or homologous chromosome (during meiosis) are used as template. Multiple layers of RNA interference exist regulating episomal and chromosomal forms of the pararetrovirus. Pararetroviruses have evolved suppressors against this plant defense in the arms race during co-evolution which can result in deregulation of plant genes. Small RNAs serve as signaling molecules for Transcriptional and Post-Transcriptional Gene Silencing (TGS, PTGS) pathways. Different populations of small RNAs comprising 21-24 nt and 18-30 nt in length have been reported for Citrus, Fritillaria, Musa, Petunia, Solanum and Beta. Recombination and RNA interference are driving forces for evolution and regulation of EPRVs.

Genetic fine-mapping of DIPLOSPOROUS in Taraxacum (dandelion; Asteraceae) indicates a duplicated DIP-gene.

BACKGROUND: DIPLOSPOROUS (DIP) is the locus for diplospory in Taraxacum, associated to unreduced female gamete formation in apomicts. Apomicts reproduce clonally through seeds, including apomeiosis, parthenogenesis, and autonomous or pseudogamous endosperm formation. In Taraxacum, diplospory results in first division restitution (FDR) nuclei, and inherits as a dominant, monogenic trait, independent from the other apomixis elements. A preliminary genetic linkage map indicated that the DIP-locus lacks suppression of recombination, which is unique among all other map-based cloning efforts of apomeiosis to date. FDR as well as apomixis as a whole are of interest in plant breeding, allowing for polyploidization and fixation of hybrid vigor, respectively. No dominant FDR or apomixis genes have yet been isolated. Here, we zoom-in to the DIP-locus by largely extending our initial mapping population, and by analyzing (local) suppression of recombination and allele sequence divergence (ASD). RESULTS: We identified 24 recombinants between two most closely linked molecular markers to DIP in an F1-population of 2227 plants that segregates for diplospory and lacks parthenogenesis. Both markers segregated c. 1:1 in the entire population, indicating a 1:1 segregation rate of diplospory. Fine-mapping showed three amplified fragment length polymorphisms (AFLPs) closest to DIP at 0.2 cM at one flank and a single AFLP at 0.4 cM at the other flank. Our data lacked strong evidence for ASD at marker regions close to DIP. An unexpected bias towards diplosporous plants among the recombinants (20 out of 24) was found. One third of these diplosporous recombinants showed incomplete penetrance of 50-85% diplospory. CONCLUSIONS: Our data give interesting new insights into the structure of the diplospory locus in Taraxacum. We postulate a locus with a minimum of two DIP-genes and possibly including one or two enhancers or cis-regulatory elements on the basis of the bias towards diplosporous recombinants and incomplete penetrance of diplospory in some of them. We define the DIP-locus to 0.6 cM, which is estimated to cover approximately 200-300 Kb, with the closest marker at 0.2 cM. Our results confirm the minor role of suppression of recombination and ASD around DIP, making it an excellent candidate to isolate via a chromosome-walking approach.

Identifying and Engineering Genes for Parthenogenesis in Plants.

Parthenogenesis is the spontaneous development of an embryo from an unfertilized egg cell. It naturally occurs in a variety of plant and animal species. In plants, parthenogenesis usually is found in combination with apomeiosis (the omission of meiosis) and pseudogamous or autonomous (with or without central cell fertilization) endosperm formation, together known as apomixis (clonal seed production). The initiation of embryogenesis in vivo and in vitro has high potential in plant breeding methods, particularly for the instant production of homozygous lines from haploid gametes [doubled haploids (DHs)], the maintenance of vigorous F1-hybrids through clonal seed production after combining it with apomeiosis, reverse breeding approaches, and for linking diploid and polyploid gene pools. Because of this large interest, efforts to identify gene(s) for parthenogenesis from natural apomicts have been undertaken by using map-based cloning strategies and comparative gene expression studies. In addition, engineering parthenogenesis in sexual model species has been investigated via mutagenesis and gain-of-function strategies. These efforts have started to pay off, particularly by the isolation of the PsASGR-BabyBoom-Like from apomictic Pennisetum, a gene proven to be transferable to and functional in sexual pearl millet, rice, and maize. This review aims to summarize the current knowledge on parthenogenesis, the possible gene candidates also outside the grasses, and the use of these genes in plant breeding protocols. It shows that parthenogenesis is able to inherit and function independently from apomeiosis and endosperm formation, is expressed and active in the egg cell, and can induce embryogenesis in polyploid, diploid as well as haploid egg cells in plants. It also shows the importance of genes involved in the suppression of transcription and modifications thereof at one hand, and in embryogenesis for which transcription is allowed or artificially overexpressed on the other, in parthenogenetic reproduction. Finally, it emphasizes the importance of functional endosperm to allow for successful embryo growth and viable seed production.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

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.