Diversity of Pol IV function is defined by mutations at the maize rmr7 locus

Abstract

Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses.

Figure 1. rmr7 encodes an NRPD2-type protein and is renamed nrpd2a.

(A) Rmr7/Nrpd2a gene model highlighting transition-type mutations in the respective mutant alleles. (B) Schematic of the predicted protein model highlighting domains (black boxes labeled A–I) that are highly conserved within all prokaryotic and eukaryotic (N)RPB2 proteins . Predicted polypeptides inferred by three mutant alleles are indicated.

Figure 2. Grasses contain multiple NRPD2-type proteins.

(A) nrpd2a and ZM2G128427 are located on homoeologous chromosome regions of 2S and 10L. Gray boxes represent maize BACs, simple sequence repeat markers, or genes. Gray lines connect homoeologous features. Chromosomes are anchored on the right by the known homoeologous gene pair of b1 and r1 . (B) Maximum likelihood tree produced from alignment (Figure S1) of NRPD2a with other plant NRPD2-like proteins. Grasses maize (prefix: ZM), sorghum (Sb), rice (Os), and Brachypodium distachyon (Bd) have multiple NRPD2-like proteins compared to eudicots poplar (Pt), grape (Vv), and Arabidopsis (At). Arabidopsis contains an additional pseudogene, nrpd2b, which is weakly expressed and does not produce a full-length protein (not shown) . NRPB2 proteins from rice and Arabidopsis root the tree. Outgroup branch length is not to scale.

Figure 3. Molecular profiles of NRPD2a action.

(A) EtBr-stained PAGE gel of small RNAs isolated from 6-day old seedlings and immature tassels of nrpd2a-1 homozygotes, nrpd1-1 homozygotes, and their respective non-mutant siblings. (B) PCR amplicons from CRM2 LTRs and the Aat gene generated from total RNAs that were random primed and treated without (−) or with (+) reverse transcriptase (RT). RNAs were isolated from 4-day old seedlings of either nrpd2a-1 homozygotes (dark seedling tissues) or heterozygous siblings (near colorless seedlings).

Figure 4. NRPD2a is required for acquisition of a B' state.

(A) One B-I Nrpd2a/b1 nrpd2a-1 plant was reciprocally crossed to one B' nrpd2a-1/B' nrpd2a-1 plant to combine B-I and B' in nrpd2a-1 homozygous mutants. (B) Resulting dark, B-I-like F₁ progeny genotypes, including both non-recombinant and recombinant types. (C) Numbers of progeny plants with the indicated color phenotypes resulting from testcrosses between thirteen F₁ plants (B) and colorless b1 Nrpd2a/b1 Nrpd2a plants. Results from individual testcrosses can be found in Table S4.