INVESTIGATION OF MOLECULAR MECHANISMS OF REGULATION OF MAIZE PERICARP COLOR1 BY AN EPIGENETIC MODIFIER UNSTABLE FACTOR FOR ORANGE1
Abstract:The pericarp color1 (p1) gene encodes an R2R3 Myb transcription factor which regulates biosynthesis of brick red phlobaphene pigments commonly seen in the pericarp and cob glumes of maize ears. A unique feature of p1 is the allelic diversity; over 100 natural alleles and epialleles have been reported most of which produce distinct tissue-specific pigmentation patterns. Alleles of p1 are classified based on pigmentation accumulation patterns of pericarp and cob glumes; P1-rr conditions red pericarp and red cob, whereas P1-wr specifies white pericarp and red cob phenotype. Diverse tissue-specific patterns displayed by p1 alleles have been attributed to their gene structure, copy number, and epigenetic mechanisms. To elucidate the role of epigenetic mechanisms in p1 allelic diversity, we have previously characterized Unstable factor for orange1 (Ufo1), a dominant modifier that disrupts the tissue-specific patterns of P1-wr, a multicopy allele hypermethylated allele of p1. P1-wr; Ufo1 plants exhibit ectopic accumulation of phlobaphenes in pericarp and other organs, and such gain of pigmentation is associated with decreased DNA methylation at p1. Furthermore, Ufo1 shows incomplete penetrance and expressivity; only a subset of P1-wr Ufo1 plants show gain of pigmentation and the heritability of pigmentation phenotype is poor. In the current study, we show that Ufo1 induces progressive hypomethylation of the P1-wr repeat complex, leading to enhanced penetrance and expressivity of the gain of pigmentation phenotype. We have also characterized P1-wr*, a hypermethylated epiallele of P1-wr, which is highly silenced and shows a white pericarp and white cob phenotype. Surprisingly, Ufo1 reactivates P1-wr* in a tissue-specific manner such that the cobs become pigmented while the pericarps remain colorless. Using extensive Southern blot analysis and genomic bisulfite sequencing, we show that cob pigmentation in P1-wr is affected by DNA methylation of a 168 bp region in the second intron. Interestingly, P1-wr*; Ufo1 plants did not show any methylation change in a distal enhancer region that has previously been implicated in Ufo1-induced gain of pericarp pigmentation of the P1-wr allele. These results suggest that distinct regulatory sequences in the P1-wr promoter and intron regions can undergo independent epigenetic modifications to generate tissue-specific expression patterns. Furthermore, results from genomic bisulfite sequencing revealed that loss of DNA methylation in P1-wr and P1-wr* was observed in CG, CNG, and CHH contexts. Since maintenance of CG and non-CG methylation is under distinct enzymatic control, this result indicates that Ufo1 factors lies upstream of DNA methylation. To test if Ufo1 is also involved in other epigenetic phenomena, we tested its role in paramutation at the p1 locus. We studied interaction of Ufo1 with two suppressed, hypermethylated epialleles of P1-rr which differ in their paramutagenic ability: P1-rr’ is paramutagenic and it heritably silences a functional P1-rr in trans while P1-prTP is a non-paramutagenic (neutral) allele. We show that Ufo1 interacts with, and reactivates both the suppressed alleles and that such reactivation is associated with DNA hypomethylation. This study indicates that DNA methylation is a shared mechanism between paramutation and epigenetic silencing, and that ufo1 is a common factor that seems to regulate these distinct epigenetic mechanisms.
INVESTIGATION OF MOLECULAR MECHANISMS OF REGULATION OF MAIZE PERICARP COLOR1 BY AN EPIGENETIC MODIFIER UNSTABLE FACTOR FOR ORANGE. GET MORE BIO-CHEMISTRY PROJECT TOPICS AND MATERIALS