Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae)

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl, endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.