Locus-specific paramutation in Zea mays is maintained by a PICKLE-like chromodomain helicase DNA-binding 3 protein controlling development and male gametophyte function

Abstract

Paramutations represent directed and meiotically-heritable changes in gene regulation leading to apparent violations of Mendelian inheritance. Although the mechanism and evolutionary importance of paramutation behaviors remain largely unknown, genetic screens in maize (Zea mays) identify five components affecting 24 nucleotide RNA biogenesis as required to maintain repression of a paramutant purple plant1 (pl1) allele. Currently, the RNA polymerase IV largest subunit represents the only component also specifying proper development. Here we identify a chromodomain helicase DNA-binding 3 (CHD3) protein orthologous to Arabidopsis (Arabidopsis thaliana) PICKLE as another component maintaining both pl1 paramutation and normal somatic development but without affecting overall small RNA biogenesis. In addition, genetic tests show this protein contributes to proper male gametophyte function. The similar mutant phenotypes documented in Arabidopsis and maize implicate some evolutionarily-conserved gene regulation while developmental defects associated with the two paramutation mutants are largely distinct. Our results show that a CHD3 protein responsible for normal plant ontogeny and sperm transmission also helps maintain meiotically-heritable epigenetic regulatory variation for specific alleles. This finding implicates an intersection of RNA polymerase IV function and nucleosome positioning in the paramutation process.

Fig 1. Mutant phenotypes.

(A) Comparison of ems063095 mutant (left) and non-mutant sibling (right) seedlings. Bar = 2cm. (B) Comparison of adult ems063095 mutant (left) and flowering non-mutant sibling (right). (C) Abaxial surface of adult ems063095 mutant leaf blade. (D) ems063095 mutant tassel at flowering. Bar = 1cm. (E) Comparison of grain set on ems98738 mutant (left) and A619 (right) generated from reciprocal crosses. Bar = 1cm.

Fig 2. rmr12 mutations disrupt a CHD3-encoding gene model.

(A) Maize gene model Zm00001d045109 highlighting transition mutations found in cDNA sequences from rmr12-1, rmr12-2, rmr12-3, and rmr12-4 mutants. (B) Predicted protein model highlighting domains diagnostic of CHD3 proteins and missense, nonsense (X), and insertion lesions corresponding to the respective transition mutations in (A). (C) Maximum likelihood tree produced from alignment of full-length maize (Zm) CHD3 protein sequences with CHD3 proteins from Arabidopsis (At) and grasses including Brachypodium distachyon (Bd), Brachypodium stacei (Bs), Oropetium thomaeum (Ot), rice (Os), Panicum hallii (Ph), Panicum virgatum (Pv), Setaria italica (Si), Setaria viridis (Sv), and Sorghum bicolor (Sb) identifies two clades (I and II). The tree is anchored with Saccharomyces cerevisiae (Sc) CHD1. Branch lengths depict substitutions per site.

Fig 3. Developmental profiles of rmr12 mutant and non-mutant F₂ siblings.

(A) Days to flowering for individual progenies segregating homozygotes for rmr12-1 (mutant n = 4, non-mutant n = 53, p = 0.0009), rmr12-2 (mutant n = 6, non-mutant n = 50, p = 5.04e-6), or rmr12-3 (mutant n = 15, non-mutant n = 98, p = 9.00e-12). (B) Plant heights at flowering for progenies described in (A), rmr12-1 (mutant n = 8, non-mutant n = 54, p = 9.86e-6), rmr12-2 (mutant n = 9, non-mutant n = 50, p = 3.89e-6), or rmr12-3 (mutant n = 15, non-mutant n = 98, p = 5.07e-22). (C) Mean internode lengths (±s.e.m.) for one progeny segregating rmr12-3 homozygotes (mutant n = 12, non-mutant n = 12). ** p<0.001, n.a. = not available (single value). (D) Mean first leaf (±s.e.m.) with adult-type leaf waxes for 9 rmr12-3 mutants and 9 non-mutants from a single progeny. * p = 0.01.

Fig 4. Morphometrics of rmr12-3 mutant and non-mutant F₂ siblings.

Mean (±s.e.m.) of leaf 6 length (A) and width (B) of individuals from two progenies at flowering (mutant n = 38, non-mutant n = 236). (C) Mean number (±s.e.m.) of lateral veins at the midpoint of leaf 10 in 7-week-old plants from three progenies (mutants n = 6, non-mutants n = 10). (D) Mean tassel branch number (±s.e.m.) of individuals from eight progenies within the A632 background. BC₃F₂ (mutant n = 17, non-mutant n = 16). BC₅F₂ (mutant n = 7, non-mutant n = 8). (E) Mean primary tassel branch length (±s.e.m.) of individuals described in (D). (F) Mean number of ear shoots (±s.e.m.) at flowering of individuals described in (D). (A) to (F) solid bars: mutant, open bars: non-mutant. n.s. = not significant (p>0.05), * p<0.05, ** p<0.001.

Fig 5. sRNA profiles in rmr12-4 mutants.

Size class distributions for all genome-mapped 18-30nt reads in Rmr12 / Rmr12 and rmr12-3 / rmr12-3 eight-day post imbibition seedlings (A) and normalized to 22nt RNA levels (B). Distribution of uniquely-mapping 18-30nt reads (C) and normalized to 22nt RNA levels (D).

Fig 6. Relative pl1 mRNA levels in rmr12 mutants.

Mean fold pl1 mRNA expression (2^-ΔΔCt) (±s.e.m) by qRT-PCR normalized to gapdh levels in biological triplicate non-mutant and rmr12-3 / rmr12-3 eight-day post imbibition seedlings. * p<0.05.

Fig 7. rmr12 mutant B1-I phenotypes.

rmr12-4 mutants displaying B-I (A) and B´ (B) states.