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. 2012 May;24(5):1761-75.
doi: 10.1105/tpc.112.097618. Epub 2012 May 4.

required to maintain repression2 is a novel protein that facilitates locus-specific paramutation in maize

Joy-El R Barbour  1 , Irene T Liao, Jennifer L Stonaker, Jana P Lim, Clarissa C Lee, Susan E Parkinson, Jerry Kermicle, Stacey A Simon, Blake C Meyers, Rosalind Williams-Carrier, Alice Barkan, Jay B Hollick
Affiliations

required to maintain repression2 is a novel protein that facilitates locus-specific paramutation in maize

Joy-El R Barbour et al. Plant Cell. 2012 May.

Abstract

Meiotically heritable epigenetic changes in gene regulation known as paramutations are facilitated by poorly understood trans-homolog interactions. Mutations affecting paramutations in maize (Zea mays) identify components required for the accumulation of 24-nucleotide RNAs. Some of these components have Arabidopsis thaliana orthologs that are part of an RNA-directed DNA methylation (RdDM) pathway. It remains unclear if small RNAs actually mediate paramutations and whether the maize-specific molecules identified to date define a mechanism distinct from RdDM. Here, we identify a novel protein required for paramutation at the maize purple plant1 locus. This required to maintain repression2 (RMR2) protein represents the founding member of a plant-specific clade of predicted proteins. We show that RMR2 is required for transcriptional repression at the Pl1-Rhoades haplotype, for accumulation of 24-nucleotide RNA species, and for maintenance of a 5-methylcytosine pattern distinct from that maintained by RNA polymerase IV. Genetic tests indicate that RMR2 is not required for paramutation occurring at the red1 locus. These results distinguish the paramutation-type mechanisms operating at specific haplotypes. The RMR2 clade of proteins provides a new entry point for understanding the diversity of epigenomic control operating in higher plants.

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Figures

Figure 1.
Figure 1.
In Vitro Transcription Analysis of Rmr2 Function. (A) Slot blots of cDNA clones from the pl1, b1, a1, and uq genes hybridized with radiolabeled nascent RNAs derived from husk nuclei isolated from mutant and nonmutant siblings. pBS is a plasmid control. (B) Quantification of mean relative transcription rates of the indicated genes normalized to uq (±se). Measurements for rmr2-1/rmr2-1 genotypes (closed bars, n = 5) are displayed relative to data of Rmr2/rmr2-1 genotypes (open bars, n = 5) set at unit value.
Figure 2.
Figure 2.
Crossing Scheme Used to Test Paramutation in the Absence of Rmr2 Function. (A) Parental genotypes and F1 characters of plants used to combine Pl′ and Pl-Rh states in rmr2-1 homozygotes. “T” refers to the T6-9 (043-1) interchange breakpoint, and “+” refers to a normal chromosome 6. Anther phenotypes are either nonmutant (Pl′) or the ACS 7 displayed by rmr2-1 mutants. Plants having the F1 genotype in bold were subjected to the test cross shown in (B). (B) Parental genotypes and test cross progeny characters used to evaluate whether or not Pl1-Rh haplotypes are transmitted from rmr2-1 mutants with the ability to facilitate paramutation (paramutagenicity). Progeny anther phenotypes are listed in Table 2.
Figure 3.
Figure 3.
r1 Paramutation Analysis. (A) Parental genotypes and F1 characters of plants used to combine specific r1 haplotypes in rmr2-1 homozygotes. Plants having the F1 genotypes in bold were subjected to the test crosses shown in (B). Plants from three distinct F1 progeny sets were evaluated. (B) Parental genotypes and test cross aleurone types used to evaluate whether or not R-r haplotypes are transmitted from rmr2-1 mutants with the ability to facilitate paramutation (paramutagenicity). (C) Histogram of mean pigment levels for kernels having mottled aleurones quantified using a reflectometer (±sd). Data represent materials generated from both Rmr2/rmr2-1 (open bars) and rmr2-1/rmr2-1 (closed bars) genotypes (n = 21, 18, 14, and 15 individual test crosses for the respective genotypes).
Figure 4.
Figure 4.
Mu Insertion Site Sequences and rmr2 Gene Model. (A) Mu insertion sites are flanked by direct sequence duplications (gray boxes). Positions of the rmr2 sequence relative to the predicted transcription start site are indicated below the sequences. The start codon is underlined. Mu insertion allele abbreviations removed the rmr2- prefix from each allele name. (B) Schematic of gene (above) and protein (below) models highlighting the four exons and conserved CTR used for phylogenetic analysis (Figure 5). Lines connecting the models mark the start, stop, and rmr2-1 nonsense codons. Relative positions of the Mu and EMS lesions are indicated. aa, amino acids.
Figure 5.
Figure 5.
RMR2 Conserved Sequence Alignments and Phylogeny. (A) Alignment of the RMR2 conserved CTR in selected multicellular plants. Each gene name is followed by the amino acid residue positions in parentheses. Residues are colored if they have >60% identity across the sampled proteins. (B) Maximum likelihood relationships based on alignments shown in (A) for representative grasses: maize (Zm), sorghum (Sorghum bicolor; Sb), rice (Oryza sativa; Os), and Brachypodium distachyon (Bd); eudicots: Aquilegia coerulea (Ac), Vitis vinifera (Vv), Manihot esculenta (Me), Populus trichocarpa (Pt), A. lyrata (Al), and A. thaliana (At); and outgroup: P. patens (Pp) and S. moellendorffii (Sm). Branch lengths correspond to the indicated bootstrap values (n = 1000). Three clades of grass proteins are labeled A, B, and C.
Figure 6.
Figure 6.
RMR2-Dependent sRNAs. (A) Ethidium bromide (EtBr) staining of PAGE fractionated sRNAs from Rmr2/rmr2-1 (+) and rmr2-1/rmr2-1 (−) siblings. Sizes of the abundant sRNA classes are indicated. nt, nucleotides. (B) Plots comparing the proportion of total, genome-matched sRNA sequences versus their size. The two graphs represent the total percentages (left) and the percentage of normalized abundances of maize-specific 22-mers found in the nonmutant samples (right). Blue and red lines correspond to the profiles from Rmr2/rmr2-1 and rmr2-1/rmr2-1 genotypes, respectively. Total genome-matched reads mapped to one or more sites in the entire maize genome. (C) Plots comparing the proportion of distinct, genome-matched sRNA sequences versus their size. The two graphs represent the total percentages (left) and the percentage of normalized abundances of maize-specific 22-mers found in the nonmutant samples (right). Blue and red lines correspond to the profiles from Rmr2/rmr2-1 and rmr2-1/rmr2-1 genotypes, respectively. Distinct genome matched reads map uniquely to the entire maize genome.
Figure 7.
Figure 7.
RMR2-Dependent Methylation of the Pl1-Rh 3′ Region. Genomic DNA samples from sibling Rmr2/rmr2-1 and rmr2-1/rmr2-1 plants (five each) digested with methylation-insensitive (BstNI; B) or -sensitive (PspGI; P) endonucleases and probed with a radiolabeled genomic clone specific to 3′ pl1 sequences.
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