DEVELOPMENTAL-CHANGES IN DNA METHYLATION OF THE 2 TOBACCO POLLEN NUCLEI DURING MATURATION

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5 -methylcytosine (anti-m(5)C) antibody and a polyclonal anti-histone H1 (antihistone) antibody as an internal standard, The specificity of th e anti-m(5)C antibody was demonstrated by a titration series against b oth single-stranded DNA and double-stranded DNA substrates in either t he methylated or unmethylated forms, The antibody was found to show si milar kinetics against both double-and single-stranded DNA, and the fl uorescence was proportional to the amount of DNA used, No signal was o bserved with unmethylated substrates, The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic d ivision that gave rise to the vegetative and generative nuclei, Howeve r, during the subsequent development of the pollen, the staining of th e generative nucleus decreased until it reached a normalized value of 1/5 of that of the vegetative nucleus, The use of a confocal microscop e makes these data independent of possible focusing artefacts. The ant i-histone antibody was used as a control to show that, while the antib ody staining directed against 5-methylcytosine changed dramatically du ring pollen maturation, the histone signal did not, We observed the ex istence of structural dimorphism amongst tobacco pollen grains, the ma jority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both s ubclasses.