Immunofluorescence staining with antisera raised against p35, a basic nuclear protein that accumulates in the pollen nuclei of Lilium longiflorum, specifically stained the nucleoli in interphase nuclei of somatic tissues, including root and leaf, and in pachytene nuclei during meiotic division, whereas antisera raised against histone H1 uniformly stained the entire chromatin domain with the exception of the nucleoli in these nuclei. Further, p35-specific antisera stained the nucleoli in root and leaf nuclei of the monocotyledonous plants Tulipa gesneriana, Allium cepa and Triticum aestivum and of the dicotyledonous plants Vicia faba and Nicotiana tabacum. Thus, these novel antisera stained the nucleoli in cells of all higher plants examined, although the staining patterns within nucleoli were somewhat different among plant species and tissues. The full-length cDNA of p35 was cloned on the basis of the partial amino acid sequence. The deduced amino acid composition and amino acid sequence of p35 indicate that this nucleolar protein is a novel variant of histone Hl. Further, p35 was strongly bound to ribosomal DNA in vitro. The results of immunoblotting of histones extracted from each tissue of the various plant species with the nucleolus-specific antibodies also suggested the conservation of similar epitope(s) in both mono- and dicotyledonous plants. From these results, it is suggested that similar variants of histone Hl are specifically distributed in the nucleoli of all plant species and help to organize the nucleolar chromatin.

We have cloned three novel histone genes using antibodies that recognize only nuclei of the male gametic (generative and sperm) cells of Lilium longiflorum. The deduced amino acid sequence of each clone shows only between 40% and 50% identity with the H2A, H2B and H3 somatic core histones of other plant species. Transcripts of these genes were first detected in bicellular pollen soon after microspore mitosis, and their mRNAs, as revealed by in situ hybridization, were observed only in the cytoplasm of the generative cells. As expression of these three genes was specific to generative cells within the bicellular pollen, we designated the clones gH2A, gH2B and gH3. Immunocytochemistry further revealed that the proteins encoded by these genes accumulated in the elongating and condensing generative nucleus during development of bicellular pollen, and were most abundant in the two sperm nuclei within an elongated pollen tube. We therefore propose that these male gamete-specific core histones contribute to chromatin condensation of male gametes or to chromatin remodeling, and result in the repression of gene expression in male gametes.