Multiple trans-sensing interactions affect meiotically heritable epigenetic states at the maize pl1 locus

Abstract

Interactions between specific maize purple plant1 (pl1) alleles result in heritable changes of gene regulation that are manifested as differences in anthocyanin pigmentation. Transcriptionally repressed states of Pl1-Rhoades alleles (termed Pl') are remarkably stable and invariably facilitate heritable changes of highly expressed states (termed Pl-Rh) in Pl'/Pl-Rh plants. However, Pl' can revert to Pl-Rh when hemizygous, when heterozygous with pl1 alleles other than Pl1-Rhoades, or in the absence of trans-acting factors required to maintain repressed states. Cis-linked features of Pl1-Rhoades responsible for these trans-sensing behaviors remain unknown. Here, genetic tests of a pl1 allelic series identify two potentially separate cis-linked features: one facilitating repression of Pl-Rh and another stabilizing Pl' in trans. Neither function is affected in ethyl-methanesulfonate-induced Pl1-Rhoades derivatives that produce truncated PL1 peptides, indicating that PL1 is unlikely to mediate trans interactions. Both functions, however, are impaired in a spontaneous Pl1-Rhoades derivative that fails to produce detectable pl1 RNA. Pl'-like states can also repress expression of a pl1-W22 allele, but this repression is not meiotically heritable. As the Pl' state is not associated with unique small RNA species representing the pl1-coding region, the available data suggest that interactions between elements required for transcription underlie Pl1-Rhoades epigenetic behaviors.

Figure 1.—

pl1-Rhoades derivative mum9515 is structurally identical to Pl1-Rhoades. (A) Southern blots were hybridized with either 5′ or 3′ pl1-specific probes (materials and methods). Approximate sizes of hybridizing fragments are indicated. (B) Restriction map of Pl1-Rhoades and probe locations based on genomic sequence (GenBank accession no. L19494). Arrows below gene structure indicate a 3′ tandem duplication. Only restriction sites generating fragments hybridizing to either the 5′ or 3′ probe are shown. E, EcoRI; H, HindIII; N, NsiI; Sa, SacI; Sp, SpeI; X, XmnI.

Figure 2.—

pl1-Rhoades loss-of-function alleles. (A) Molecular lesions of pl1-Rhoades(ems9718), ems9711, and ems9703. Exons (thick boxes), translation start site, and positions of lesions are relative to +1 as defined by Cone et al. (1993b). Solid arrows indicate positions of sequencing primers; open arrows indicate positions of primers used for RT–PCR reactions (*, the forward primer used for anther RNA RT–PCR; ^‡, the forward primer used for seedling RT–PCR; see materials and methods). (B) RT–PCR of Pl1-Rhoades and two derivative alleles from anther RNA. The notations 316 bp and 426 bp are the sizes of properly spliced and unspliced pl1 transcript, respectively. H₂O designates the negative control in the PCR reaction.

Figure 3.—

pl1-Rhoades(mum9515) is an RNA null. Reverse transcriptase–PCR analysis of seedling RNA. pl1: spliced, 171 bp; unspliced, 281 bp. aat: spliced, 290 bp; unspliced, 454 bp (not shown). Negative controls: −RNA, full assay in absence of RNA; H₂O, no template in PCR stage only.

Figure 4.—

pl1-W22 is sensitive to dosage and paramutagenic pl1 alleles. (A) Crosses, progeny, and diagnostic pollen phenotypes used to expose pl1-W22 to both Pl′ and pl1-Rhoades(ems9710)′ states. (B) Representative anthers of the given genotypes collected from single mature florets at the beginning of dehiscence.

Figure 5.—

All pl1 alleles produce sense-oriented small RNAs. (A) Map of pl1-transcribed region, partial cDNA probe, and location of oligo controls. (B) Northern blot of husk tissue small RNAs from isogenic Pl-Rh/Pl-Rh and Pl′/Pl′ plants. AS (antisense), S (sense). (C) Northern blot of seedling small RNAs from pl1-CO159, pl1-B73, and pl1-W22 homozygotes. Ethidium-bromide-stained tRNA for the relevant samples is shown below blots in B and C as a loading control. See materials and methods for additional details.