In order to maintain genome integrity from generation to generation, transposons and repetitive DNA elements must be kept under tight regulation in reproductive cells. One of the ways that plants achieve this is through the stable inheritance of DNA methylation. Plants frequently show meiotic inheritance of gene silencing (1
). Furthermore, plants are not known to undergo genome-wide waves of demethylation in germ cells as occurs in animals. However, large scale reprogramming occurs in non-germ line reproductive cells, and this reprogramming may function to reinforce silencing of transposable elements in germ cells (see below).
One way to actively reprogram the epigenome is to remove methylated cytosines. The Arabidopsis genome encodes four bifunctional helix-hairpin-helix DNA glycosylases / AP lyases, REPRESSOR OF SILENCING 1 (ROS1), DEMETER (DME), DEMETER-LIKE 2 (DML2), and DEMETER-LIKE 3 (DML3), which recognize and remove methylated cytosines, resulting in single-nucleotide gap in the DNA double strand. Subsequently, as yet unidentified DNA repair polymerase and DNA ligase enzymes are thought to fill in the gap with an unmethylated cytosine (1
). ROS1, DML2, and DML3 mainly function in vegetative tissues, and genomic studies suggest that they demethylate hundreds of specific loci across the genome with a bias toward genes (1
). Knocking out all three genes does not dramatically affect the overall levels or patterns of methylation in Arabidopsis genome (18
). Instead, these enzymes appear to be acting as counterbalance to the RNA-directed DNA methylation system to quantitatively fine tune methylation levels at particular genomic locations.
By contrast, DME functions to cause global hypomethylation in the endosperm (the extra-embryonic tissue of flowering plants) of Arabidopsis (20
), and thus contributes to large scale epigenetic reprogramming (). In Arabidopsis, female gametogenesis begins when a somatically derived megaspore mother cell undergoes meiosis to give rise to a haploid megaspore, which subsequently develops into a mature female gametophyte (embryo sac) that contains one egg cell, one central cell (two nuclei), and several other accessory cells. During double fertilization (which is normal in plants), the egg cell fuses with a sperm cell from male gametophyte (pollen grain) to form an embryo, and the central cell fuses with the other sperm cell from pollen to form the triploid endosperm, which nourishes the embryo, and thus bears a similar function as the placenta of mammals. DME is expressed primarily in the central cell before fertilization, and thus only the maternal genome is demethylated by DME. This leads to maternal allele specific gene expression (imprinting) in the endosperm (22
). Until recently, only six imprinted Arabidopsis genes were known but recent genomic studies of endosperm have revealed genome-wide differences in DNA methylation including a dramatic reduction of CG methylation, showing that many additional genes are likely to be imprinted in Arabidopsis, some of which have been verified by single gene studies (20
) (). Demethylation by DME may also reactivate transposon expression, which shunts transposon transcripts into the RNAi pathway, producing additional siRNAs that can guide DNA methylation to non-CG sites whose methylation is high in wild-type endosperm but decreased in dme
mutant endosperm (). Curiously, there are even higher levels of non-CG methylation in the wild-type embryo which could be explained by movement of siRNAs produced in the central cell into the egg cell, an attractive idea that is waiting for experimental support (20
). Because the endosperm genome does not contribute to the next generation, mild reactivation of transposons in endosperm may not be deleterious, and may reinforce the silencing of transposons in the egg cell and later embryo, contributing to genome integrity of offspring. Indeed there is a class of Pol IV-dependent siRNAs that only accumulates in flowers and young siliques, likely originating from the endosperm (23
). Notably, these siRNAs are derived from maternal alleles only, suggesting that they may be produced in part during female gametogenesis and then retained after karyogamy. However, these siRNAs are expressed more highly after fertilization, suggesting that imprinted maternal expression of siRNA loci also takes place as the endosperm develops (23
). It is tempting to speculate that the maternal Pol IV-dependent siRNAs are the “messenger” that mediates communication between endosperm and embryo (), however this is not supported by the finding that these siRNAs were only detected in endosperm not embryo (23
). Nevertheless, the possibility that they exist in low abundance in the embryo, or are ephemeral, cannot be ruled out.
The model that siRNAs move from the endosperm to the embryo is consistent with the model put forth by an earlier study on paternal genome reprogramming in Arabidopsis (24
). The male gametophyte of Arabidopsis (a pollen grain) contains two sperm cells, which fertilize the egg cell and central cell, respectively, and a vegetative nucleus (). Transposon expression is generally upregulated in pollen, and certain transposons even become mobile in pollen, contrasting with the situation in most other tissues (24
). Reduction of transposon methylation and their reactivation appears to occur in the vegetative nucleus, which is supported by the fact that transposon reactivation and movement is not inherited by the next generation (24
). It has been shown that several key RdDM pathway proteins (RDR2 and DCL3) and CHG methylation maintenance pathway proteins (CMT3 and KYP) have reduced expression levels in pollen, and in addition DECREASE IN DNA METHYLATION 1 (DDM1), an important chromatin remodeler required for DNA and histone methylation and transposon silencing, is exclusively localized in sperm cells but not in the vegetative nucleus (24
). These results suggest a model in which hypomethylation of the vegetative cell may reactivate transposons that could serve to reinforce transposon silencing in the adjacent sperm cells () (24
Small RNAs may be involved in this communication between the vegetative cell and the sperm cells. A class of siRNAs that is 21 nucleotides in length and corresponds to Athila
retrotransposons, the largest transposon family, is detected in sperm cells. Since Athila
retrotransposons remain silenced in sperm cells but are activated in the vegetative nucleus, this supports the idea that the 21-nt siRNAs are produced in the vegetative nucleus and then travel to their site of action – sperm cells-- where they mediate the silencing of transposons through a yet unknown mechanism () (24
). A common theme is that both male and female gametophytes contain nurse cells in which massive epigenetic reprogramming may serve to reinforce transposon silencing in the germline ().
Another example of small RNAs silencing transposons at a distance occurs when the megaspore mother cell differentiates from somatic tissues (25
). Mutations in ARGONAUTE 9 (AGO9), a member of the Arabidopsis Argonaute family of proteins, result in the reactivation of transposons in the ovule (including the egg cell), in which AGO9
expression is not detected (). Remarkably, AGO9
is not expressed in the reproductive cells themselves (megaspore mother cell, megaspore, or developing female gametophyte), but in the companion cells surrounding the female gametophyte. Notably, the transposon targets of AGO9 are similar to those reactivated in pollen and evidence suggests that AGO9-mediated transposon silencing utilizes components of known silencing pathways including the 24-nt RNA-directed DNA methylation pathway (25
). Whether the AGO9-associated 24-nt siRNAs are the mobile signal remains to be tested.