The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Small RNAs from plants are known to be highly complex and abundant, with this complexity proportional to genome size. Most endogenous siRNAs in Arabidopsis are dependent on RNA-DEPEN-DENT RNA POLYMERASE 2 (RDR2) for their biogenesis. Recent work has demonstrated that the maize MEDIATOR OF PARAMUTATION1 (mop1) gene is a predicted ortholog of RDR2. The mop1 gene is required for establishment of paramutation and maintenance of transcriptional silencing of transposons and transgenes, suggesting the potential involvement of small RNAs. We analyzed small RNAs in wild-type maize and in the isogenic mop1-1 loss-offunction mutant by using Illumina's sequencing-by-synthesis (SBS) technology, which allowed us to characterize the complement of maize small RNAs to considerable depth. Similar to rdr2 in Arabidopsis, in mop1-1, the 24-nucleotide (nt) endogenous heterochromatic short-interfering siRNAs were dramatically reduced, resulting in an enrichment of miRNAs and transacting siRNAs. In contrast to the Arabidopsis rdr2 mutant, the mop1-1 plants retained a highly abundant heterochromatic Ϸ22-nt class of small RNAs, suggesting a second mechanism for heterochromatic siRNA production. The enrichment of miRNAs and loss of 24-nt heterochromatic siRNAs in mop1-1 should be advantageous for miRNA discovery as the maize genome becomes more fully sequenced.miRNA ͉ mop1 ͉ rdr2 ͉ small RNA T he small RNAs found in a typical plant cell include a small number of highly abundant, mainly 21-nt microRNAs (miRNAs) and a large number of small interfering RNAs (mainly 24 nt heterochromatic siRNAs, or simply siRNAs) recognizing many diverse sequences. In addition, several additional subclasses of varying and in some cases overlapping functional importance have been described, including the transacting siRNAs (ta-siRNAs) (1-3), natural antisense siRNAs, a type thus far observed only under stress conditions (4, 5), and natural antisense miRNAs (6).MicroRNAs have a variety of regulatory roles in development and stress responses (for review, see ref. 7). In addition, work in Arabidopsis has led to the hypothesis that transcription of repeats is performed by the plant-specific RNA polymerase IV (pol IV) followed by reverse transcription and cleavage by RDR2 and DCL3, respectively. These repeated sequences include transposons and retrotransposons in plants (8), and this series of events produces a complex set of heterochromatic siRNAs (for review, see ref. 9). The genome size of plants varies substantially among species mainly caused by variation in content of repeated DNA. The complexity of siRNAs is correspondingly greater in rice than in Arabidopsis (10), consistent with larger numbers of repeated sequences in rice.The maize b1 locus is an excellent model for paramutation, a phenomenon in which alleles communicate in trans, resulting in meiotically heritable gene expression changes (11). Molecular work, combined with fine-structure recombination mapping, has demonstrated that this activity is mediated by tandem repeats at b1 that are required to e...

Whether deposited maternal products are important during early seed development in flowering plants remains controversial. Here, we show that RNA interference-mediated downregulation of transcription is deleterious to endosperm development but does not block zygotic divisions. Furthermore, we show that RNA POLYMERASE II is less active in the embryo than in the endosperm. This dimorphic pattern is established late during female gametogenesis and is inherited by the two products of fertilization. This juxtaposition of distinct transcriptional activities correlates with differential patterns of histone H3 lysine 9 dimethylation, LIKE HETEROCHROMATIN PROTEIN1 localization, and Histone H2B turnover in the egg cell versus the central cell. Thus, distinct epigenetic and transcriptional patterns in the embryo and endosperm are already established in their gametic progenitors. We further demonstrate that the non-CG DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and DEMETER-LIKE DNA glycosylases are required for the correct distribution of H3K9 dimethylation in the egg and central cells, respectively, and that plants defective for CMT3 activity show abnormal embryo development. Our results provide evidence that cell-specific mechanisms lead to the differentiation of epigenetically distinct female gametes in Arabidopsis thaliana. They also suggest that the establishment of a quiescent state in the zygote may play a role in the reprogramming of the young plant embryo.

Paramutation involves homologous sequence communication that leads to meiotically heritable transcriptional silencing. We demonstrate that mop2 (mediator of paramutation2), which alters paramutation at multiple loci, encodes a gene similar to Arabidopsis NRPD2/E2, the second-largest subunit of plant-specific RNA polymerases IV and V. In Arabidopsis, Pol-IV and Pol-V play major roles in RNA–mediated silencing and a single second-largest subunit is shared between Pol-IV and Pol-V. Maize encodes three second-largest subunit genes: all three genes potentially encode full length proteins with highly conserved polymerase domains, and each are expressed in multiple overlapping tissues. The isolation of a recessive paramutation mutation in mop2 from a forward genetic screen suggests limited or no functional redundancy of these three genes. Potential alternative Pol-IV/Pol-V–like complexes could provide maize with a greater diversification of RNA–mediated transcriptional silencing machinery relative to Arabidopsis. Mop2-1 disrupts paramutation at multiple loci when heterozygous, whereas previously silenced alleles are only up-regulated when Mop2-1 is homozygous. The dramatic reduction in b1 tandem repeat siRNAs, but no disruption of silencing in Mop2-1 heterozygotes, suggests the major role for tandem repeat siRNAs is not to maintain silencing. Instead, we hypothesize the tandem repeat siRNAs mediate the establishment of the heritable silent state—a process fully disrupted in Mop2-1 heterozygotes. The dominant Mop2-1 mutation, which has a single nucleotide change in a domain highly conserved among all polymerases (E. coli to eukaryotes), disrupts both siRNA biogenesis (Pol-IV–like) and potentially processes downstream (Pol-V–like). These results suggest either the wild-type protein is a subunit in both complexes or the dominant mutant protein disrupts both complexes. Dominant mutations in the same domain in E. coli RNA polymerase suggest a model for Mop2-1 dominance: complexes containing Mop2-1 subunits are non-functional and compete with wild-type complexes.

Bryophytes (liverworts, hornworts and mosses) comprise the three earliest diverging lineages of land plants (embryophytes). Marchantia polymorpha, a complex thalloid Marchantiopsida liverwort that has been developed into a model genetic system, occupies a key phylogenetic position. Therefore, M. polymorpha is useful in studies aiming to elucidate the evolution of gene regulation mechanisms in plants. In this study, we used computational, transcriptomic, small RNA and degradome analyses to characterize microRNA (miRNA)-mediated pathways of gene regulation in M. polymorpha. The data have been integrated into the open access ContigViews-miRNA platform for further reference. In addition to core components of the miRNA pathway, 129 unique miRNA sequences, 11 of which could be classified into seven miRNA families that are conserved in embryophytes (miR166a, miR390, miR529c, miR171-3p, miR408a, miR160 and miR319a), were identified. A combination of computational and degradome analyses allowed us to identify and experimentally validate 249 targets. In some cases, the target genes are orthologous to those of other embryophytes, but in other cases, the conserved miRNAs target either paralogs or members of different gene families. In addition, the newly discovered Mpo-miR11707.1 and Mpo-miR11707.2 are generated from a common precursor and target MpARGONAUTE1 (LW1759). Two other newly discovered miRNAs, Mpo-miR11687.1 and Mpo-miR11681.1, target the MADS-box transcription factors MpMADS1 and MpMADS2, respectively. Interestingly, one of the pentatricopeptide repeat (PPR) gene family members, MpPPR_66 (LW9825), the protein products of which are generally involved in various steps of RNA metabolism, has a long stem–loop transcript that can generate Mpo-miR11692.1 to autoregulate MpPPR_66 (LW9825) mRNA. This study provides a foundation for further investigations of the RNA-mediated silencing mechanism in M. polymorpha as well as of the evolution of this gene silencing pathway in embryophytes.

SummaryParamutation involves trans interactions between alleles or homologous sequences that establish distinct gene expression states that are heritable for generations. It was first described in maize by Alexander Brink in the 1950's, with his studies of the r1 (red1) locus. Since that time, paramutation-like phenomena have been reported in other maize genes, other plants, fungi and animals. Paramutation can occur between endogenous genes, two transgenes or an endogenous gene and transgene. Recent results indicate that paramutation involves RNA-mediated heritable chromatin changes and a number of genes implicated in RNAi pathways. However, not all aspects of paramutation can be explained by known mechanisms of RNAi-mediated transcriptional silencing. This study represents a milestone in plant biology. The sequence of the highly repetitive maize genome, one of the most important crop plants for humanity and a classical biological research model.

Paramutation is the epigenetic transfer of information between alleles that leads to the heritable change of expression of one allele. Paramutation at the b1 locus in maize requires seven noncoding tandem repeat (b1TR) sequences located ∼100 kb upstream of the transcription start site of b1, and mutations in several genes required for paramutation implicate an RNA-mediated mechanism. The mediator of paramutation (mop1) gene, which encodes a protein closely related to RNA-dependent RNA polymerases, is absolutely required for paramutation. Herein, we investigate the potential function of mop1 and the siRNAs that are produced from the b1TR sequences. Production of siRNAs from the b1TR sequences depends on a functional mop1 gene, but transcription of the repeats is not dependent on mop1. Further nuclear transcription assays suggest that the b1TR sequences are likely transcribed predominantly by RNA polymerase II. To address whether production of b1TR-siRNAs correlated with paramutation, we examined siRNA production in alleles that cannot undergo paramutation. Alleles that cannot participate in paramutation also produce b1TR-siRNAs, suggesting that b1TR-siRNAs are not sufficient for paramutation in the tissues analyzed. However, when b1TR-siRNAs are produced from a transgene expressing a hairpin RNA, b1 paramutation can be recapitulated. We hypothesize that either the b1TR-siRNAs or the dsRNA template mediates the trans-communication between the alleles that establishes paramutation. In addition, we uncovered a role for mop1 in the biogenesis of a subset of microRNAs (miRNAs) and show that it functions at the level of production of the primary miRNA transcripts.interchromosomal | transfer | epigenetic | information | trans-generational

Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.