mediator of paramutation1 is required for establishment and maintenance of paramutation at multiple maize loci

Abstract

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.

Figure 1.

Phenotypes Associated with the mop1-1 Mutation. (A) B′ Mop1/mop1-1. (B) B′ mop1-1/mop1-1. (C) B-I Mop1/Mop1. (D) B′ mop1-1/mop1-1 plant with B′-like sectors. (E) Pl′ Mop1/- (either Mop1/Mop1 or Mop1/mop1-1). (F) Pl′ mop1-1/mop1-1.

Figure 2.

Diagram Outlining Test for Heritability of B′ and Pl′ from Homozygous mop1-1 Individuals. (A) If mop1-1 heritably alters B′ to B-I, then B′ mop1-1/mop1-1 individuals would generate B-I mop1-1 gametes and progeny would be dark. Alternately, if B′ is still B′, then all gametes would be B′ mop1-1, B′ would paramutate B-I (indicated by [B-I]′) in the next generation, and all progeny would be light. (B) If mop1-1 heritably alters Pl′ to Pl-Rh, then Pl′ mop1-1/mop1-1 individuals would generate Pl-Rh mop1-1 gametes and progeny would have dark anthers. Alternately, if Pl′ is still Pl′, then all gametes would be Pl′ mop1-1, Pl′ would paramutate Pl-Rh (indicated by [Pl-Rh]′) in the next generation, and all progeny would have light anthers.

Figure 3.

ACSs of Pl′ mop1-1/mop1-1 Outcrosses versus Pl′ Mop1/mop1-1 Outcrosses. Pl′ mop1-1/mop1-1 individuals versus Pl′ Mop1/mop1-1 individuals were outcrossed with Pl-Rh (Mop1/Mop1) testers. Individual progeny plants were scored for amount of anther pigment.

Figure 4.

Amounts of Transcripts in mop1-1 versus Wild-Type Siblings. An example of RNase protections for pl1, b1, and actin1 on four sibling individuals. All individuals are homozygous B′ and Pl′ and segregate for mop1-1 as indicated. The bar graph shows the normalized amounts of b1 (open bars) and pl1 (closed bars) RNA from the RNase protection.

Figure 5.

Transcription Rates in mop1-1 versus Wild-Type Siblings. (A) An example of an in vitro transcription assay showing the SK+ plasmid negative control and the signal for b1, c2, and ubiquitin2 transcription in B′ mop1-1/mop1-1 versus B′ Mop1/mop1-1 individuals. (B) Paired data for b1 transcription rate from several in vitro transcription assays for several mop1-1/mop1-1 (closed bars) versus Mop1/mop1-1 (open bars) individuals. The data represent three separate comparisons between a pair of sibling individuals. The n-fold (designated X) increase is given below each pairwise comparison. (C) Paired data from RNase protection assays for b1 RNA quantities normalized to quantities of ubiquitin2 RNA for the same mop1-1/mop1-1 (closed bars) versus Mop1/mop1-1 (open bars) sibling individuals as examined in (B).

Figure 6.

Diagram Showing Three Progeny Classes from B′/B-Peru Mop1/mop1-1 Self-Pollinations. B′ mop1-1 plants were crossed with B-Peru Mop1 plants. The light F₁ B′/B-Peru Mop1/mop1-1 plants were self-pollinated, and purple kernels (B-Peru/-) were planted. The progeny fell into three phenotypic classes; the numbers and genotypes of each phenotype are shown. The mop1 genotype of the 24 B-Peru/B-Peru plants was determined by test crosses with B′ mop1-1.

Figure 7.

Diagram Outlining Test for Ability of mop1-1 to Prevent b1 Paramutation. B′/B′ mop1-1/mop1-1 plants were crossed with B-I/b Mop1/mop1-1 plants, generating four types of segregating progeny. The B′/B-I and B′/b progeny were distinguished by restriction fragment length polymorphisms. The B′/b progeny were not analyzed further. Both classes of B′/B-I progeny (dark and light) were crossed with B-I/B-Peru Mop1/Mop1 testers (and with testers null for b1; not diagrammed) to test whether the B-I allele heterozygous with B′ had become B′ in the mop1-1/mop1-1 versus Mop1/mop1-1 plants. The expectations for crosses with B′/B-I mop1-1/mop1-1 with one type of tester (B-Peru Mop1, which gives purple kernels) are shown. The expectation for the B′/B-I Mop1/mop1-1 progeny is that all offspring will be light plants (not diagrammed).

Figure 8.

Summary of mop1-1 Effects on r1 Paramutation. (A) Parental genotypes and progeny classes used to evaluate the effect of mop1-1 on the establishment of r1 paramutation. mop1 genotypes were determined on the basis of the intensity of the vegetative tissue pigment within the Pl/- phenotypic class. Individuals belonging to the two R-d/- genotypic classes were identified by crossing with an r-g tester stock, and the R-d/r-g kernel progeny were then assayed for pigment intensity. (B) The bar graph shows results of paramutation test with r1. Color scores for kernels inheriting the R-d allele are plotted from individuals having the parental genotype indicated along the x axis. The first two columns are Mop1/- genotype controls. The color score equals 100 minus the average reflectometer reading, as described by Alleman and Kermicle (1993). Error bars indicate sd.

Figure 9.

Phenotypes Characteristic of mop1 Mutations Relative to Wild-Type Siblings. (A) A B′ Mop1/mop1-1 individual bearing a normal tassel. (B) A B′ mop1-1/mop1-1 individual bearing a feminized tassel (strong tasselseed). (C) A B′/- mop1-2EMS/mop1-2EMS individual bearing a severely barrenized tassel. (D) A runty B′ mop1-1/mop1-1 individual in which the feminized terminal inflorescence failed to emerge.

Figure 10.

DNA Gel Blots Assaying Methylation of Repeated Sequences. Individual genotypes are indicated above the DNA gel blots (B' stands for B′/B′ Mop1/Mop1). (A) Samples digested with the methylation-insensitive enzyme BstNI (B) and the methylation-sensitive enzyme EcoRII (E) were probed with the 45S ribosomal repeat. (B) Samples digested with HpaII (H) or MspI (M) were probed with the centromere repeat.