Jane E. Dorweiler

An RNA-dependent RNA polymerase is required for paramutation in maize

Paramutation is an allele-dependent transfer of epigenetic information, which results in the heritable silencing of one allele by another. Paramutation at the b1 locus in maize is mediated by unique tandem repeats that communicate in trans to establish and maintain meiotically heritable transcriptional silencing. The mop1 (mediator of paramutation1) gene is required for paramutation, and mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Here we report the map-based cloning of mop1, an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. Nuclear run-on assays reveal that the tandem repeats required for b1 paramutation are transcribed from both strands, but siRNAs were not detected. We propose that the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.

Teosinte glume architecture 1: A genetic locus controlling a key step in maize evolution

Teosinte, the probable progenitor of maize, has kernels that are encased in hardened fruitcases, which interfere with the use of the kernels as food. Although the components of the fruitcase are present in maize, their development is disrupted so that the kernels are not encased as in teosinte but exposed on the ear. The change from encased to exposed kernels represents a key step in maize evolution. The locus that largely controls this morphological difference between maize and teosinte, teosinte glume architecture 1, is described and genetically mapped.

mediator of paramutation1 Is Required for Establishment and Maintenance of Paramutation at Multiple Maize Loci

Paramutation is the directed, heritable alteration of the expression of one allele when heterozygous with another allele. Here, the isolation and characterization of a mutation affecting paramutation, mediator of paramutation1-1 (mop1-1), are described. Experiments demonstrate that the wild-type gene Mop1 is required for establishment and maintenance of the paramutant state. The mop1-1 mutation affects paramutation at the multiple loci tested but has no effect on alleles that do not participate in paramutation. The mutation does not alter the amounts of actin and ubiquitin transcripts, which suggests that the mop1 gene does not encode a global repressor. Maize plants homozygous for mop1-1 can have pleiotropic developmental defects, suggesting that mop1-1 may affect more genes than just the known paramutant ones. The mop1-1 mutation does not alter the extent of DNA methylation in rDNA and centromeric repeats. The observation that mop1 affects paramutation at multiple loci, despite major differences between these loci in their gene structure, correlations with DNA methylation, and stability of the paramutant state, suggests that a common mechanism underlies paramutation. A protein-based epigenetic model for paramutation is discussed.

Paramutation in maize

Paramutation is a heritable change in gene expression induced by allele interactions. This review summarizes key experiments on three maize loci, which undergo paramutation. Similarities and differences between the phenomenology at the three loci are described. In spite of many differences with respect to the stability of the reduced expression states at each locus or whether paramutation correlates with DNA methylation and repeated sequences within the loci, recent experiments are consistent with a common mechanism underlying paramutation at all three loci. Most strikingly, trans-acting mutants have been isolated that prevent paramutation at all three loci and lead to the activation of silenced Mutator transposable elements. Models consistent with the hypothesis that paramutation involves heritable changes in chromatin structure are presented. Several potential roles for paramutation are discussed. These include localizing recombination to low-copy sequences within the genome, establishing and maintaining chromatin domain boundaries, and providing a mechanism for plants to transmit an environmentally influenced expression state to progeny.

Paramutation and related allelic interactions

Paramutation is an allelic interaction that results in meiotically heritable changes in gene expression. Until recently, the few documented cases in higher plants seemed unusual and rare. This perception is rapidly fading because of the discovery of related examples and the growing recognition of epigenetic changes in a wide variety of biological systems.

Developmental analysis of teosinte glume architecture1: A key locus in the evolution of maize (Poaceae).

A key event in the evolution of maize from teosinte was a reduction in the cupulate fruitcase and softening of the glumes, which increased the accessibility of kernels for harvest. The teosinte glume architecture1 (tga1) locus largely controls this difference between maize and teosinte, and thus may have played a pivotal role in maize evolution. The teosinte allele (tga1+teosinte) lengthens inflorescence internodes, shortens rachillae, and makes glumes longer, thicker, and harder. Developmental characterization of morphometric traits reveals that differences among genotypes are apparent early in female inflorescence development. Increased hardening in glumes homozygous for tga1+teosinte is correlated with a thicker abaxial mesoderm of lignified cells. Silica deposition in the abaxial epidermal cells of the glumes is also affected. In the maize background, glumes homozygous for tga1+teosinte deposit silica in both the short and long cells of the glume epidermis, whereas glumes homozygous for the maize allele (Tga1+Maize) concentrate silica only in the short cells. Silica deposition also appears to be affected by genetic background. The effects of tga1 appear largely to explain the differences in glume induration between maize and teosinte. The diverse pleiotropic effects of tga1 suggest that it is regulatory in nature.

A mutation that prevents paramutation in maize also reverses Mutator transposon methylation and silencing

Both paramutation and Mutator (Mu) transposon inactivation involve heritable changes in gene expression without concomitant changes in DNA sequence. The mechanisms by which these shifts in gene activity are achieved are unknown. Here we present evidence that these two phenomena are linked mechanistically. We show that mutation of a gene, modifier of paramutation 1 (mop1), which prevents paramutation at three different loci in maize, can reverse methylation of Mutator elements reliably. In mop1 mutant backgrounds, methylation of nonautonomous Mu elements can be reversed even in the absence of the regulatory MuDR element. Previously silenced MuDR elements are reactivated sporadically after multiple generations of exposure to mop1 mutations. MuDR methylation is separable from MuDR silencing, because removal of methylation does not cause immediate reactivation. The mop1 mutation does not alter the methylation of certain other transposable elements including those just upstream of a paramutable b1 gene. Our results suggest that the mop1 gene acts on a subset of epigenetically regulated sequences in the maize genome and paramutation and Mu element methylation require a common factor, which we hypothesize influences chromatin structure.

A Dominant Mutation in mediator of paramutation2, One of Three Second-Largest Subunits of a Plant-Specific RNA Polymerase, Disrupts Multiple siRNA Silencing Processes

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.

A maize CONSTANS-like gene, conz1, exhibits distinct diurnal expression patterns in varied photoperiods

Maize (Zea mays ssp. mays L.) was domesticated from teosinte (Z. mays L. ssp. parviglumis Iltis & Doebley), a plant requiring short day photoperiods to flower. While photoperiod sensitive landraces of maize exist, post-domestication breeding included efforts to grow maize in a broad range of latitudes. Thus, modern maize is often characterized as day-neutral because time to flower is relatively unaffected by photoperiod. We report the first identification of maize constans of Zea mays1 (conz1), a gene with extensive sequence homology to photoperiod genes CONSTANS (CO) in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) and Heading date1 (Hd1) in rice (Oryza sativa L.). conz1 maps to a syntenous chromosomal location relative to Hd1. Additionally, conz1 and two maize homologs of another photoperiod gene exhibit diurnal expression patterns notably similar to their Arabidopsis and rice homologs. The expression pattern of conz1 in long days is distinct from that observed in short days, suggesting that maize is able to discern variations in photoperiod and respond with differential expression of conz1. We offer models to reconcile the differential expression of conz1 with respect to the photoperiod insensitivity exhibited by temperate inbreds.

Repeat associated small RNAs vary among parents and following hybridization in maize

Small RNAs (sRNAs) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73 and Mo17) and their hybrid. We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in siRNAs primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared with Arabidopsis or rice, we confirmed that, like these simpler plant genomes, 24-nt siRNAs whose abundance differs between maize parents also show a trend of down-regulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase 2 encoded by modifier of paramutation1 (mop1). We also discovered that 21–22-nt siRNAs derived from a number of distinct retrotransposon families differentially accumulate between B73 and Mo17 as well as their hybrid. Thus, maize possesses a unique source of genetic variation for regulating transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis.