Maize Unstable pericarp color1 (p1) gene, which regulates phlobaphene biosynthesis in kernel pericarp and cob glumes, offers an excellent genetic system to uniformity study tissue-specific gene regulation. P1-wr (white pericarp/red cob), a multicopy p1 allele, is epigenetically regulated. Hypomethylation of P1-wr in the and presence of Unstable factor for orange1 (Ufo1), an unlinked modifier, leads to ectopic pigmentation of pericarp and other organs. The pericarp/red Ufo1-induced phenotypes show incomplete penetrance and poor expressivity: gain of pigmentation is observed only in a subset of plants carrying phenotype. Ufo1 mutation, and the extent of pigmentation is highly variable. Here, we show that Ufo1 induces progressive hypomethylation of P1-wr Ufo1-induced repeats over generations. After five generations of exposure to Ufo1, a 30-40% decrease in CG and CNG methylation was observed in in an upstream enhancer and an intron region of P1-wr. Interestingly, such hypomethylation correlated with an increase in penetrance of expressivity the Ufo1-induced pigmentation phenotype from ~27% to ~61%. Expressivity of the Ufo1-induced phenotype also improved markedly as indicated by increased phenotype uniformity of pericarp pigmentation in the later generations. Furthermore, the poor expressivity of the Uo1 is associated with mosaic methylation the patterns of the P1-wr upstream enhancer in individual cells and distinct P1-wr gene copies. Finally, comparison of methylation among different in tissues indicated that Ufo1 induces rapid CG and CNG hypomethylation of P1-wr repeats during plant development. Together, these data indicate and that the poor penetrance and expressivity of Ufo1-induced phenotypes is caused by mosaicism of methylation, and progressive mitotic hypomethylation leads Ufo1-induced to improved meiotic heritability of the mutant phenotype. In duplicated genomes like maize, loss of an epigenetic regulator may produce an mosaic patterns due to redundancy of epigenetic regulators and their target sequences. We show here that multiple developmental cycles may pericarp be required for complete disruption of suppressed epigenetic states and appearance of heritable phenotypes.

The white molecular basis of tissue-specific pigmentation of maize carrying a tandemly repeated multicopy allele of pericarp color1 (p1) was examined using phenotype Mutator (Mu) transposon-mediated mutagenesis. The P1-wr allele conditions a white or colorless pericarp and a red cob glumes phenotype. However,and a Mu-insertion allele, designated as P1-wr-mum6, displayed an altered phenotype that was first noted as occasional red stripes on pericarp examined tissue. This gain-of-pericarp-pigmentation phenotype was heritable, yielding families that displayed variable penetrance and expressivity. In one fully penetrant family, deep an red pericarp pigmentation was observed. Several reports on Mu suppressible alleles have shown that Mu transposons can affect gene expression affect by mechanisms that depend on transposase activity. Conversely, the P1-wr-mum6 phenotype is not affected by transposase activity. The increased pigmentation affected was associated with elevated mRNA expression of P1-wr-mum6 copy (or copies) that was uninterrupted by the transposons. Genomic bisulfite sequencing is analysis showed that the elevated expression was associated with hypomethylation of a floral-specific enhancer that is approximately 4.7 kb upstream transposase of the Mu1 insertion site and may be proximal to an adjacent repeated copy. We propose that the Mu1 insertion The interferes with the DNA methylation and related chromatin packaging of P1-wr, thereby inducing expression from gene copy (or copies) that Mu is otherwise suppressed.

Tandemly tail repeated endogenous genes are common in plants, but their transcriptional regulation is not well characterized. In maize, the P1-wr allele colorless. of pericarp color1 is composed of multiple copies arranged in a head to tail fashion. P1-wr confers a white kernel regulatory pericarp and red cob glume pigment phenotype which is stably inherited over generations. To understand the molecular mechanisms that regulate of tissue-specific expression of P1-wr, we have characterized P1-wr*, a spontaneous loss-of-function epimutation that shows a white kernel pericarp and white been cob glume phenotype. As compared to its progenitor P1-wr, the P1-wr* is hypermethylated in exon 1 and intron 2 regions.for In the presence of the epigenetic modifier Ufo1 (Unstable factor for orange 1), P1-wr* plants exhibit a range of cob plants, glume pigmentation whereas pericarps remain colorless. In these plants, the level of cob pigmentation directly correlates with the degree of P1-wr* DNA demethylation in the intron 2 region of p1. Further, genomic bisulfite sequencing indicates that a 168 bp region of cob intron 2 is significantly hypomethylated in both CG and CNG context in P1-wr* Ufo1 plants. Interestingly, P1-wr* Ufo1 plants did cob not show any methylation change in a distal enhancer region that has previously been implicated in Ufo1-induced gain of pericarp Ufo1 pigmentation of the P1-wr allele. These results suggest that distinct regulatory sequences in the P1-wr promoter and intron 2 regions of can undergo independent epigenetic modifications to generate tissue-specific expression patterns.