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Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Publication . Calarco, Joseph P.; Borges, Filipe; Donoghue, Mark T.A.; Van Ex, Frédéric; Jullien, Pauline E.; Lopes, Telma; Gardner, Rui; Berger, Frédéric; Feijó, José A.; Becker, Jörg D.; Martienssen, Robert A.

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

2012-09-28Journal article

Restricted access

Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signature

Publication . Becker, Jörg D; Takeda, Seiji; Borges, Filipe; Dolan, Liam; Feijó, José A

Current views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants.

2014-08-01Journal article

Open access

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei

Publication . Borges, Filipe; Gardner, Rui; Lopes, Telma; Calarco, Joseph P; Boavida, Leonor C; Slotkin, R; Martienssen, Robert A; Becker, Jörg D

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

2012-10-17Journal article

Open access

Arabidopsis Tetraspanins Are Confined to Discrete Expression Domains and Cell Types in Reproductive Tissues and Form Homo- and Heterodimers When Expressed in Yeast

Publication . Boavida, L. C.; Qin, P.; Broz, M.; Becker, J. D.; McCormick, S.

Tetraspanins are evolutionary conserved transmembrane proteins present in all multicellular organisms. In animals, they are known to act as central organizers of membrane complexes and thought to facilitate diverse biological processes, such as cell proliferation, movement, adhesion, and fusion. The genome of Arabidopsis (Arabidopsis thaliana) encodes 17 members of the tetraspanin family; however, little is known about their functions in plant development. Here, we analyzed their phylogeny, protein topology, and domain structure and surveyed their expression and localization patterns in reproductive tissues. We show that, despite their low sequence identity with metazoan tetraspanins, plant tetraspanins display the typical structural topology and most signature features of tetraspanins in other multicellular organisms. Arabidopsis tetraspanins are expressed in diverse tissue domains or cell types in reproductive tissues, and some accumulate at the highest levels in response to pollination in the transmitting tract and stigma, male and female gametophytes and gametes. Arabidopsis tetraspanins are preferentially targeted to the plasma membrane, and they variously associate with specialized membrane domains, in a polarized fashion, to intercellular contacts or plasmodesmata. A membrane-based yeast (Saccharomyces cerevisiae) two-hybrid system established that tetraspanins can physically interact, forming homo- and heterodimer complexes. These results, together with a likely genetic redundancy, suggest that, similar to their metazoan counterparts, plant tetraspanins might be involved in facilitating intercellular communication, whose functions might be determined by the composition of tetraspanin complexes and their binding partners at the cell surface of specific cell types.

2013-10Journal article

Open access

Evolutionarily conserved mechanisms of male germline development in flowering plants and animals

Publication . Pereira, Patrícia A.; Navarro-Costa, Paulo; Martinho, Rui Gonçalo; Becker, Jörg D.

Sexual reproduction is the main reproductive strategy of the overwhelming majority of eukaryotes. This suggests that the last eukaryotic common ancestor was able to reproduce sexually. Sexual reproduction reflects the ability to perform meiosis, and ultimately generating gametes, which are cells that carry recombined half sets of the parental genome and are able to fertilize. These functions have been allocated to a highly specialized cell lineage: the germline. Given its significant evolutionary conservation, it is to be expected that the germline programme shares common molecular bases across extremely divergent eukaryotic species. In the present review, we aim to identify the unifying principles of male germline establishment and development by comparing two very disparate kingdoms: plants and animals. We argue that male meiosis defines two temporally regulated gene expression programmes: the first is required for meiotic commitment, and the second is required for the acquisition of fertilizing ability. Small RNA pathways are a further key communality, ultimately ensuring the epigenetic stability of the information conveyed by the male germline.

2014-04-01Journal article

Open access