Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Summary

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Abstract

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal 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 cell 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, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Protocol

I. Genetic Crossing

1. Emasculating the female parent

In order to distinguish the maternal and paternal alleles by using DNA sequence polymorphism, two different ecotypes, e.g. Columbia-0 (Col-0) and Ler, will be chosen as female and male parents. The plants should be young and healthy. One can emasculate the female parent by using a dissecting microscope, magnifying visor, or naked eye. Locate stage-12 flowers (Smyth et al., 1990) and remove any flowers or siliques above and below them by clipping the base of the pedicel with the scissors. Sterilize forceps by dipping the base of the tip gently into a beak....

Discussion

It is relatively easy to separate embryo from endosperm and seed coat, but it is tedious to separate endosperm from seed coat, especially for seed at early or mid-torpedo stage of embryogenesis. Since seed coat only contributes a very small amount of tissue, for some genes, e.g., MEA and FWA, we do not have to separate endosperm from seed coat. That means that we can isolate RNAs from a mixture of endosperm and seed coat tissues, check expression of the maternal and paternal alleles of a gene, compare w.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Supplies Dissecting Microscope Scissors Fine Tip Forceps Jewelry Tag Plant Stakes String or Twist-Ties 4" X 2" X 8" Polyethylene Bags 3" X 1" X 1.0 mm Microscope Slides Liquid Nitrogen Liquid Nitrogen Containers Heat Block PCR Tubes Thermocycler Microcentrifuge Tubes Microcentrifuge Gel Electrophoresis facility Arabidopsis thaliana Columbia-0 Plants Arabidopsis thaliana Landsberg erecta Plants Reagents 70% Ethanol 95% Ethanol 100% Ethanol 0.3 M Sorbitol and 5 mM MES-pH 5.7 Cetyltrimethyl Ammonium Bromide (CTAB) 100% Ethanol Cholorform Restriction Enzymes 3 M NaOH 6.3 M NaOH 6.24 M Urea/ 4 M Sodium Bisulfite Sterile distilled H2O 10 mM Hydroquinone Wizard DNA Clean-Up System (Promega) 10 M NH4OAc 20 μg/ μL tRNA TE buffer The TOPO TA Cloning Kit (Invitrogen)