Journal Article

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Podesta, A, Jost, J.P, Oakeley, E.J

1997

Overview

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone HI (antihistone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of 1/2 of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses

• Publisher: National Academy of Sciences of the United States of America,National Acad Sciences,National Academy of Sciences,The National Academy of Sciences of the USA
• Subject: 5-METHYLCYTOSINE / ADN / analogs & derivatives / analysis / Antibodies / Antibody Specificity / ANTICORPS MONOCLONAL / ANTICUERPOS MONOCLONALES / BINDING / Biological Sciences / Botany / Cell Nucleus / Cell Nucleus - physiology / Cell Nucleus - ultrastructure / chemistry / CITOSINA / cytology / CYTOSINE / Cytosine - analogs & derivatives / Cytosine - analysis / Daughter cells / Deoxyribonucleic acid / DNA / DNA Methylation / DNA, Plant / DNA, Plant - metabolism / DOUBLE STRANDED DNA / Fluorescence / Fluorescence microscopy / GAMETOGENESE / GAMETOGENESIS / GENERATIVE NUCLEUS / growth & development / HISTONAS / HISTONE / HISTONES / IMMUNOCYTOCHEMISTRY / IMMUNOLOGIE / IMMUNOLOGY / INMUNOLOGIA / Meiosis / metabolism / Methylation / Microscopy, Confocal / MITOSE / MITOSIS / MONOCLONAL ANTIBODIES / Nicotiana - growth & development / NICOTIANA TABACUM / NOYAU CELLULAIRE / NUCLEO / NUCLEUS / Oligodeoxyribonucleotides / Oligodeoxyribonucleotides - chemistry / Oligonucleotides / physiology / Plants / Plants, Toxic / POLEN / POLLEN / Pollen - cytology / Pollen - physiology / POLLEN DEVELOPMENT / SINGLE STRANDED DNA / Somatic cells / Tobacco / ultrastructure / VEGETATIVE NUCLEUS