Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

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

Tissue-specific patterns of a maize Myb transcription factor are epigenetically regulated

The maize p1 gene encodes a Myb-homologous regulator of red pigment biosynthesis. To investigate the tissue-specific regulation of the p1 gene, maize plants were transformed with constructs combining promoter and cDNA sequences of two alleles which differ in pigmentation patterns: P1-wr (white pericarp/red cob) and P1-rr (red pericarp/red cob). Surprisingly, all promoter/cDNA combinations produced transgenic plants with red pericarp and red cob (RR pattern), indicating that the P1-wr promoter and encoded protein can function in pericarp. Some of the RR patterned transgenic plants produced progeny plants with white pericarp and red cob (WR pattern), and this switch in tissue-specificity correlated with increased transgene methylation. A similar inverse correlation between pericarp pigmentation and DNA methylation was observed for certain natural p1 alleles, which have a gene structure characteristic of standard P1-wr alleles, but which confer red pericarp pigmentation and are consistently less methylated than standard P1-wr alleles. Although we cannot rule out the possible existence of tissue-specific regulatory elements within the p1 non-coding sequences or flanking regions, the data from transgenic and natural alleles suggest that the tissue-specific pigmentation pattern characteristic of the P1-wr phenotype is epigenetically controlled.     

A Mutator transposon insertion is associated with ectopic expression of a tandemly repeated multicopy Myb gene pericarp color1 of maize

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

Ac Induces Homologous Recombination at the Maize P Locus

The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences.     

Maize Unstable factor for orange1 Is Required for Maintaining Silencing Associated with Paramutation at the pericarp color1 and booster1 Loci

To understand the molecular mechanisms underlying paramutation, we examined the role of Unstable factor for orange1 (Ufo1) in maintaining paramutation at the maize pericarp color1 (p1) and booster1 (b1) loci. Genetic tests revealed that the Ufo1-1 mutation disrupted silencing associated with paramutation at both p1 and b1. The level of up regulation achieved at b1 was lower than that at p1, suggesting differences in the role Ufo1-1 plays at these loci. We characterized the interaction of Ufo1-1 with two silenced p1 epialleles, P1-rr′ and P1-pr^TP, that were derived from a common P1-rr ancestor. Both alleles are phenotypically indistinguishable, but differ in their paramutagenic activity; P1-rr′ is paramutagenic to P1-rr, while P1-pr^TP is non-paramutagenic. Analysis of cytosine methylation revealed striking differences within an enhancer fragment that is required for paramutation; P1-rr′ exhibited increased methylation at symmetric (CG and CHG) and asymmetric (CHH) sites, while P1-pr^TP was methylated only at symmetric sites. Both silenced alleles had higher levels of dimethylation of lysine 9 on histone 3 (H3K9me2), an epigenetic mark of silent chromatin, in the enhancer region. Both epialleles were reactivated in the Ufo1-1 background; however, reactivation of P1-rr′ was associated with dramatic loss of symmetric and asymmetric cytosine methylation in the enhancer, while methylation of up-regulated P1-pr^TP was not affected. Interestingly, Ufo1-1–mediated reactivation of both alleles was accompanied with loss of H3K9me2 mark from the enhancer region. Therefore, while earlier studies have shown correlation between H3K9me2 and DNA methylation, our study shows that these two epigenetic marks are uncoupled in the Ufo1-1–reactivated p1 alleles. Furthermore, while CHH methylation at the enhancer region appears to be the major distinguishing mark between paramutagenic and non-paramutagenic p1 alleles, H3K9me2 mark appears to be important for maintaining epigenetic silencing.     

Effect of flavonoid pigments on the accumulation of fumonisin B1 in the maize kernel

Mycotoxins are secondary metabolites with potential dangers for animal and human health. In particular, maize (Zea mays L.) infection caused by Fusarium and the consequent fumonisin contamination is widespread in several countries such as Italy. We developed six maize populations differing in their constitution of regulatory genes able to accumulate respectively anthocyanins in the aleurone layer (r1 gene), pericarp (b1 and pl1 genes) and phlobaphene in the pericarp (p1 gene). These coloured populations, with the related control colourless populations were analysed for mycotoxin content in the kernels during three field seasons with the aim of understanding if there were any correlations with their ability to accumulate flavonoids in kernel tissues. Our results indicate that accumulation of flavonoid pigments in the seeds, in particular phlobaphenes, is able to reduce the level of fumonisin B1. This finding could be used to minimize kernel mycotoxin contamination in this crop, in particular, the development of sweet, pop and polenta coloured corn varieties will help the farmer to keep the level of fumonisin under the threshold of contamination established for human corn consumption.     

Elements of the maize A1 promoter required for transactivation by the anthocyanin B/C1 or phlobaphene P regulatory genes.

The extensive genetic and molecular characterization of the flavonoid pathway's structural and regulatory genes has provided some of the most detailed knowledge of gene interactions in plants. In maize flavonoid biosynthesis, the A1 gene is independently regulated in the anthocyanin and phlobaphene pathways. Anthocyanin production requires the expression of the C1 or PI and R or B regulatory genes, whereas phlobaphene production requires only the P regulatory gene. By deletion analysis of the A1 promoter, we show that the sequences between -123 and -88 are critical for activation by anthocyanin and phlobaphene regulatory genes. Linker-scanner mutations indicated that the -123 to -100 region is more important for transactivation by the P protein. The -98 to -88 region is more important for B/C1 transactivation and shows a strong homology with the region of the Bz1 anthocyanin structural gene promoter shown to be activated by B/C1 and not by P. We identified a 14-bp consensus sequence that is also present in the promoters of three other genes in the anthocyanin pathway, and we propose a model for how the flavonoid regulatory proteins interact with the promoters of the structural genes.