Small RNA-mediated DNA methylation during plant reproduction

Abstract

Reproductive tissues are a rich source of small RNAs, including several classes of short interfering (si)RNAs that are restricted to this stage of development. In addition to RNA polymerase IV-dependent 24-nt siRNAs that trigger canonical RNA-directed DNA methylation, abundant reproductive-specific siRNAs are produced from companion cells adjacent to the developing germ line or zygote and may move intercellularly before inducing methylation. In some cases, these siRNAs are produced via non-canonical biosynthesis mechanisms or from sequences with little similarity to transposons. While the precise role of these siRNAs and the methylation they trigger is unclear, they have been implicated in specifying a single megaspore mother cell, silencing transposons in the male germ line, mediating parental dosage conflict to ensure proper endosperm development, hypermethylation of mature embryos, and trans-chromosomal methylation in hybrids. In this review, we summarize the current knowledge of reproductive siRNAs, including their biosynthesis, transport, and function.

Figure 1

Male reproductive development, siRNA biogenesis, and proposed intercellular movement. A, Male germ line development. A microspore mother cell undergoes meiosis to generate four haploid microspores (shown in tetrad stage). Each microspore has an asymmetric mitotic division to create the vegetative cell and the generative cell. The generative cell later divides to create the two sperm cells. Together, the vegetative cell and sperm cells form the male gametophyte or mature pollen grain. Microspore mother cells, microspores, and immature pollen are surrounded by a layer of sporophytic cells called the tapetum or tapetal nurse cells. B, The Arabidopsis tapetum produces 24-nt nurse cell siRNAs through the canonical RdDM pathway. Pol IV and CLSY3 produce RNA precursors which are converted into dsRNA by RDR2 before processing by DCL3 to generate 24-nt nurse cell siRNAs. Nurse-cell siRNAs move into microspore mother cells, inducing DNA methylation with the aid of DRM1 and/or DRM2 (DRM). C, In the tapetum of many other angiosperms, reproductive phasiRNAs are produced from PHAS loci. Pol II transcribes PHAS loci to generate precursor transcripts, which are targeted by miRNAs for cleavage. The cleaved transcripts are converted into dsRNA by RDR6, and those targeted by miR2275 are further processed by DCL5 to generate 24-nt phasiRNAs. These phasiRNAs move intercellularly into germ cells, and might induce DNA methylation (left arrow) or post-transcriptional gene silencing through transcript cleavage (right arrow). D, Epigenetically-activated (ea)siRNAs are produced from reactivated transposons in the vegetative nucleus, where DME and ROS1 actively demethylate transposons and some protein coding genes. The demethylated loci are transcribed by either Pol II or Pol IV and the resulting RNAs are cleaved by miRNA/AGO1 complexes, triggering their conversion into dsRNA by RDR2 or/and RDR6. DCL2 and DCL4 then produce 22- and 21-nt easiRNAs from the dsRNA, respectively. These vegetative cell-derived easiRNAs can induce gene silencing in the sperm cell, either through transcriptional gene silencing (left arrow) or post-transcriptional gene silencing (right arrow).

Figure 2

Female reproductive development and the action of maternal 24-nt reproductive siRNAs. A, Female germ line development. Within each ovule, a single MMC undergoes meiosis to generate four haploid megaspores. Three of these degenerate, leaving a single functional megaspore. The megaspore goes through three rounds of nuclear division before cytokinesis to generate a 7-celled female gametophyte (antipodal cells not shown). The female gametophyte contains a binucleate central cell and a haploid egg cell that are ready for fertilization. The female germ line is surrounded by somatic cells throughout this development. B, Before meiosis, Pol IV, RDR2, and DCL3 produce 24-nt siRNAs, which interact with AGO9 to mediate DNA methylation and transcriptional gene silencing of SPL/NZZ via DRM1 and/or DRM2 (DRM). Regulation of SPL/NZZ is required for the specification of a single MMC. C, In somatic cells of a mature ovule, CLSY3 and CLSY4 direct Pol IV to transcribe siren loci. These transcripts are converted into double stranded by RDR2 and further processed into 24-nt siren siRNAs by DCL3. siren siRNAs induce methylation at protein coding genes in somatic cells and are proposed to move intercellularly, causing methylation in the gametophyte.

Figure 3

24-nt siRNA production during seed development. A, The developing seed is composed of three tissues, with distinct genetic complements. m, maternal or matrigenic; p, patrigenic. B, Pol IV activity in the central cell is hypothesized to establish an epigenetic state that is maintained on matrigenic chromosomes in the endosperm (orange halo). This epigenetic state causes allele-specific siRNA production in the endosperm and might also influence imprinted gene expression. However, whether methylation caused by allele-specific siRNAs reinforces allele-specific gene expression is unclear. (MEG, PEG; filled arrows depict the expressed state while hollow arrows with dashed outline show the non-expressed allele). C, Siren siRNAs are produced in the immature seed coat and trigger DNA methylation at protein-coding genes via DRM proteins. Siren siRNAs might also move intercellularly, resulting in maternally specific accumulation of siRNAs in the endosperm. Siren siRNA methylation of protein-coding genes in the endosperm might influence seed development. D, During embryo development, canonical RdDM is upregulated at many transposons, resulting in hypermethylation of the genome in mature embryos. This methylation is rapidly lost upon germination.

Figure 4

Trans-chromosomal methylation and demethylation. Four hypothetical loci (A–D) that are differentially methylated between two varieties. At loci A and C, there is no allelic interaction, and methylation depends on the genetic background (green is methylated, purple is unmethylated). At locus B, TCM triggers methylation on the previously unmethylated allele (orange hexagon). In repeated backcrosses to the unmethylated background, the newly methylated allele continues to induce methylation of naive alleles. At locus D, TCdM results in demethylation of the high methylation alleles (grey dashed hexagon). This unmethylated state continues to cause TCdM in subsequent generations of backcrossing to the methylated allele.