Specialized technologies for epigenetics in plants.

Plants are excellent systems for discovering and studying epigenetic phenomena, such as transposon silencing, RNAi, imprinting, and DNA methylation. Imprinting, referring to preferential expression of maternal or paternal alleles, plays an important role in reproduction development of both mammals and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA (MEA), a SET domain Polycomb group gene, was the first plant gene shown to be imprinted in endosperm, and its maternal expression is controlled by DNA methylation and demethylation. Recently there has been significant progress in identifying imprinted genes as well as understanding molecular mechanisms of imprinting in plants. Up to date, approximately 350 genes were found to have differential parent-of-origin expression in plant endosperm (Arabidopsis, corn, and rice). In Arabidopsis, many imprinted genes are regulated by the DNA METHYLTRANSFERASE1 (MET1) and the DNA-demethylating glycosylase DEMETER (DME), and/or their chromatin states regulated by Polycomb group proteins (PRC2). There are also maternally expressed genes regulated by unknown mechanisms in endosperm. In this protocol, we describe in detail how to perform a genetic cross, isolate the endosperm tissue from seed, determine the imprinting status of a gene, and analyze DNA methylation of imprinted genes by bisulfite sequencing in Arabidopsis.