Histones and DNA ultrastructural distribution in plant cell nucleus: a combination of immunogold and cytochemical methods.

In this work we report for the first time the ultrastructural distribution of histones and DNA in the nuclear compartments in two different plant cell types: Allium cepa L. root meristems and Capsicum annuum L. microspores and pollen grains, by using antibodies against histones H2B and H4 and anti-DNA. Immunolocalizations were combined with ultrastructural cytochemistry for nucleic acids (methylation-acetylation method), DNA (NAMA-Ur) and RNPs (EDTA), to relate the subcellular location of histones and DNA with the chemical subcompartmentalization of the cell nucleus. This is particularly interesting concerning the presence of histones or not on fibers of the interchromatin region and on the fibrillar components of the nucleolus, nuclear subcompartments where transcription has been shown to take place at some regions. Our methodological approach permitted to define precisely the structures where histones were detected in relation to the ultrastructural localization of chromatin in various structural condensation levels. Concerning the localization of DNA and histones on the different components of the nucleolus, the combination of immunogold labeling with the methylation-acetylation cytochemical method, developed in our laboratory, was very useful, thus permitting a clear recognition of the nucleolar components and a correct assignment of labeling, which is not always evident on uranyl-lead-stained Lowicryl sections. Double immunogold assays were also done for a simultaneous visualization of histones and DNA. Our results show a coincident distribution of histones and DNA on the same nuclear compartments revealing the presence of both antigens on condensed chromatin, fibers of the interchromatin region, principally located at the periphery of the condensed chromatin, and in the fibrillar components of the nucleolus.