Summary The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that would transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with down-regulation of the heterochromatin remodeler DECREASE IN DNA METHYLATION 1 and of most TE siRNAs. However, 21 nucleotide siRNA from Athila retrotransposons is generated in pollen and accumulates in sperm, indicating that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes.

Plant genomes produce a variety of small RNAs that function in distinct, yet overlapping, genetic and epigenetic silencing pathways. However, the abundance and diversity of small RNA classes varies in different plant species, suggesting co-evolution between environmental adaptations and gene silencing mechanisms. Small RNA biogenesis in plants is well understood, but we are just beginning to uncover their intricate regulation and activity. Here, we discuss the biogenesis of plant small RNAs, such as microRNAs, secondary small-interfering RNAs and heterochromatic small-interfering RNAs, and their diverse cellular and developmental functions, including reproductive transitions, genomic imprinting and paramutation. We also discuss the diversification of small RNA-directed silencing pathways through the expansion of RNA-dependent RNA polymerases, Dicer and Argonaute proteins.

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.NIH grant: (R01 GM067014); Temasek Lifescience Laboratory; NSERC graduate student fellowship; Fred C. Gloeckner Foundation; Belgian American Educational Foundation postdoctoral fellowship: (Herbert Hoover)

In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE1 (DCL1)-dependent miRNAs, that also trigger 21-nt secondary siRNA via RNA DEPENDENT RNA POLYMERASE6 (RDR6), DCL4, and ARGONAUTE1 (AGO1)1–3, while transcriptional gene silencing (TGS) of transposons is mediated by 24-nt heterochromatic (het)siRNA RDR2, DCL3 and AGO44. Transposons can also give rise to abundant 21-nt “epigenetically activated” small interfering RNAs (easiRNAs) in DECREASE IN DNA METHYLATION1 (ddm1) and DNA METHYLTRANSFERASE1 (met1) mutants, as well as in the vegetative nucleus of pollen grains5, and in dedifferentiated plant cell cultures6. Here we show that easiRNAs resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than fifty miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nt easiRNA, and severe infertility, but 24-nt hetsiRNA are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germline. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing, and by their hosts for genome defence.

Elevations in cytosolic free calcium concentration ([Ca(2+)](cyt)) constitute a fundamental signal transduction mechanism in eukaryotic cells, but the molecular identity of Ca(2+) channels initiating this signal in plants is still under debate. Here, we show by pharmacology and loss-of-function mutants that in tobacco and Arabidopsis, glutamate receptor-like channels (GLRs) facilitate Ca(2+) influx across the plasma membrane, modulate apical [Ca(2+)](cyt) gradient, and consequently affect pollen tube growth and morphogenesis. Additionally, wild-type pollen tubes grown in pistils of knock-out mutants for serine-racemase (SR1) displayed growth defects consistent with a decrease in GLR activity. Our findings reveal a novel plant signaling mechanism between male gametophyte and pistil tissue similar to amino acid-mediated communication commonly observed in animal nervous systems.

Eukaryotic centromeres contain the kinetochore, which connects chromosomes to the spindle allowing segregation. During meiosis, centromeres are suppressed for inter-homolog crossover, as recombination in these regions can cause chromosome missegregation and aneuploidy. Plant centromeres are surrounded by transposon-dense pericentromeric heterochromatin that is epigenetically silenced by histone 3 lysine 9 dimethylation (H3K9me2), and DNA methylation in CG and non-CG sequence contexts. However, the role of these chromatin modifications in control of meiotic recombination in the pericentromeres is not fully understood. Here, we show that disruption of Arabidopsis thaliana H3K9me2 and non-CG DNA methylation pathways, for example, via mutation of the H3K9 methyltransferase genes KYP/SUVH4 SUVH5 SUVH6, or the CHG DNA methyltransferase gene CMT3, increases meiotic recombination in proximity to the centromeres. Using immunocytological detection of MLH1 foci and genotyping by sequencing of recombinant plants, we observe that H3K9me2 and non-CG DNA methylation pathway mutants show increased pericentromeric crossovers. Increased pericentromeric recombination in H3K9me2/non-CG mutants occurs in hybrid and inbred backgrounds and likely involves contributions from both the interfering and noninterfering crossover repair pathways. We also show that meiotic DNA double-strand breaks (DSBs) increase in H3K9me2/non-CG mutants within the pericentromeres, via purification and sequencing of SPO11-1-oligonucleotides. Therefore, H3K9me2 and non-CG DNA methylation exert a repressive effect on both meiotic DSB and crossover formation in plant pericentromeric heterochromatin. Our results may account for selection of enhancer trap Dissociation (Ds) transposons into the CMT3 gene by recombination with proximal transposon launch-pads.

Chromosome dosage plays a significant role in reproductive isolation and speciation in both plants and animals, but underlying mechanisms are largely obscure1. Transposable elements can promote hybridity through maternal small RNA2, and have been postulated to regulate dosage response via neighboring imprinted genes3,4. Here, we show that a highly conserved microRNA in plants, miR845, targets the tRNAMet primer-binding site (PBS) of LTR-retrotransposons in Arabidopsis pollen, and triggers the accumulation of 21 to 22-nucleotide small RNA in a dose dependent fashion via RNA polymerase IV. We show that these epigenetically activated small-interfering RNAs (easiRNAs) mediate hybridization barriers between diploid seed parents and tetraploid pollen parents (“the triploid block”), and that natural variation for miR845 may account for “endosperm balance” allowing formation of triploid seeds. Targeting the PBS with small RNA is a common mechanism for transposon control in mammals and plants, and provides a uniquely sensitive means to monitor chromosome dosage and imprinting in the developing seed.