Abstract
Two proteins that resemble core histones and might be specific to the male gametic (generative) nucleus within the pollen of Lilium longiflorum Thumb, (originally designated p22.5 and p18.5; K. Ueda and I. Tanaka, 1994, Planta, 192, 446–452) were characterized biochemically and immunochemically. Patterns of digestion of p22.5 and p18.5 by Staphylococcus aureus V8 protease closely resembled those of somatic histones H2B and H3, respectively. However, peptide fragments that were unique to p22.5 or p18.5 were also detected. Antibodies raised against these proteins did not cross-react with any somatic histones. These results indicate that p22.5 and p18.5 are different from somatic histones in terms of primary structure. Analysis of their amino-acid compositions revealed that p22.5 is a moderately lysine-rich protein while p18.5 is an arginine-rich protein. From these results, we conclude that p22.5 is a variant of histone H2B and p18.5 is a variant of histone H3. Immunofluorescence staining of pollen grains using the specific antibodies revealed that both p22.5 and p18.5 are only present in the generative cell nucleus and are not to be found in the vegetative cell nucleus. This study demonstrates that (i) specific histone variants are present in the male gametic nucleus of a higher plant, as they are in the sperm nucleus of animals, and (ii) distinct differences in histone composition exist between the nuclei of generative and vegetative cells in pollen. These novel histones (p22.5 and p18.5), specific to male gametic nuclei, have been designated gH2B and gH3, respectively.
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Abbreviations
- DAPI:
-
4′6-diamidino-2-phenylindole
- FITC:
-
fluorescein isothiocyanate
References
Allis, C.D., Glover, C.V.C., Gorovsky, M.A. (1979) Micronuclei of Tetrahymena contain two types of histone H3. Proc. Natl. Acad. Sci. USA 76, 4857–4861
Allis, C.D., Glover, C.V.C., Bowen, J.K., Gorovsky, M.A. (1980) Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eukaryote, Tetrahymena thermophila. Cell 20, 609–617
Blomstedt, C.K., Xu, H., Singh, M.B., Knox, R.B. (1992) The isolation and purification of surface specific proteins of somatic and reproductive protoplasts of lily and rapeseed. Physiol. Plant. 85, 396–402
Chiu, M.L., Irvin, J.L. (1984) Isolation of rat testis histone TH2B-x, and interaction of TH2B-x antiserum with histones and mononucleosomes. Arch. Biochem. Biophys. 229, 295–303
Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K. (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102–1106
Hasenkampf, C., Qureshi, M., Horsch, A., Riggs, C.D. (1992) Temporal and spatial distribution of meiotin-1 in anthers of Lilium longiflorum. Dev. Gen. 13, 425–434
Hough, T., Singh, M.B., Smart, I.J., Knox, R.B. (1986) Immunofluorescent screening of monoclonal antibodies to surface antigens of animal and plant cells bound to polycarbonate membranes. J. Immunol. Methods 92, 103–107
Isenberg, I. (1979) Histones. Annu. Rev. Biochem. 48, 159–191
Kasinsky, H.E. (1989) Specificity and distribution of sperm basicproteins. In: Histones and other basic nuclear proteins, pp. 73–163, Hnilica, L.S., ed. CRC Press, Boca Raton, Fla.
Knox, R.B., Zee, S.Y., Blomstedt, C., Singh, M.B. (1993) Male gametes and fertilization in angiosperms. New Phytol. 125, 679–694
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Luna, E.J., Kidd, G.H., Branton, D. (1979) Identification by peptide analysis of the spectrin-binding protein in human erythrocytes. J. Biol. Chem. 254, 2526–2532
McGhee, J.D., Felsenfeld, G. (1980) Nucleosome structure. Annu. Rev. Biochem. 49, 1115–1156
Meistrich, M.L. (1989) Histone and basic nuclear protein transitions in mammalian spermatogenesis. In: Histones and other basic nuclear proteins, pp. 166–182, Hnilica, L.S., ed. CRC Press, Boca Raton, Fla.
Meistrich, M.L., Bucci, L.R., Trostle-Weige P.K., Brock W.A. (1985) Histone variants in rat spermatogonia and primary spermatocytes. Dev. Biol. 112, 230–240
Oliva, R., Dixon, G.H. (1991) Vertebrate protamine genes and the histone-to-protamine replacement reaction. Progr. Nucleic. Acid Res. Mol. Biol. 40, 25–94
Pennell, R.I., Geltz, N.R., Koren, E., Russell, S.D. (1987) Production and partial characterization of hybridoma antibodies elicited to the sperm of Plumbago zeylanica. Bot. Gaz. 148, 401–406
Poccia, D. (1986) Remodeling of nucleoproteins during gametogene sis, fertilization, and early development. Int. Rev. Cytol. 105, 1–65
Poccia, D.L., Simpson, M.V., Green, G.R. (1987) Transitions in histone variants during sea urchin spermatogenesis. Dev. Biol. 121, 445–453
Reynolds, W.F., Wolfe, S.L. (1978) Changes in basic proteins during sperm maturation in a plant, Marchantia polymorpha. Exp. Cell Res. 116, 269–273
Reynolds, W.F., Wolfe, S.L. (1984) Protamines in plant sperm. Exp. Cell Res. 152, 443–448
Riggs, C.D., Hasenkampf, C.A. (1991) Antibodies directed against a meiosis-specific, chromatin-associated protein identify conserved meiotic epitopes. Chromosoma 101, 92–98
Rodrigo, M.I., Franco, L. (1990) Histone variants from pea (Pisum sativum): Their differential presence in fractions obtained by DNase I digestion of nuclei. Physiol. Plant. 78, 602–608
Rodrigues, J.D.A., Brandt, W.F., Von Holt, C. (1979) Plant histone 2 from wheat germ, a family of histone H2A variants: Partial amino acid sequences. Biochim. Biophys. Acta 578, 96–206
Sasaki, Y., Harada, H. (1991) Binding form of pollen mother cell protein in the nucleosomes of lily. Plant Physiol. 96, 1161–1166
Sasaki, Y., Yasuda, H., Ohba, Y., Harada, H. (1990) Isolation and characterization of a novel nuclear protein from pollen mother cells of lily. Plant Physiol. 94, 1467–1471
Seyedin, S.M., Kistler, W.S. (1980) Isolation and characterization of rat testis H1t. J. Biol. Chem. 255, 5949–5954
Sheridan, W.F., Stern, H. (1967) Histones of meiosis. Exp. Cell Res. 45, 323–335
Shires, A., Carpenter, M.P. (1976) A cysteine-containing H2B-like histone found in mature mammalian testis. J. Biol. Chem. 251, 4155–4158
Spiker, S. (1988) Histone variants and high mobility group nonhistone chromosomal proteins of higher plants: Their potential for forming a chromatin structure that is either poised for transcription or transcriptionally inert. Physiol. Plant. 75, 200–213
Spiker, S., Krishnaswamy, L. (1973) Constancy of wheat histones during development. Planta 110, 71–76
Spiker, S., Key, J.L., Wakim, B. (1976) Identification and fractionation of plant histones. Arch. Biochem. Biophys. 176, 510–518
Takahashi, T., Ikai, A., Takahashi, K. (1989) Purification and characterization of proline-β-naphthylamidase, a novel enzyme from pig intestinal mucosa. J. Biol. Chem. 264, 11565–11571
Tanaka, I., Kitazume, C., Ito, M. (1987) The isolation and culture of lily pollen protoplasts. Plant Sci. 50, 205–211
Tanaka, I., Nakamura, S., Miki-Hirosige, H. (1989) Structural features of isolated generative cells and their protoplasts from pollen of some liliaceous plants. Gamete Res. 24, 361–374
Theunis, C.H., Pierson, E.S., Cresti, M. (1991) Isolation of male and female gametes in higher plants. Sex. Plant Reprod. 4, 145–154
Trostle-Weige, P.K., Meistrich, M.L., Brock, W.A., Nishioka, K. Bremer, J.W. (1982) Isolation and characterization of TH2A, a germ cell-specific variant of histone 2A in rat testis. J. Biol. Chem. 257, 5560–5567
Trostle-Weige, P.K., Meistrich, M.L., Brock, W.A., Nishioka, K. (1984) Isolation and characterization of TH3, a germ cell-specific variant of histone 3 in rat testis. J. Biol. Chem. 259, 8769–8776
Ueda, K., Tanaka, I. (1994) The basic proteins of male gametic nuclei isolated from pollen grains of Lilium longiflorum. Planta 192, 446–452
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The authors thank Dr. Y. Sado (Shigei Medical Institute, Japan) for his helpful advice on immunization and Prof. T. Iguchi and Prof. K. Manabe (Yokohama City University, Japan) for providing facilities for experiments. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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Ueda, K., Tanaka, I. Male gametic nucleus-specific H2B and H3 histones, designated gH2B and gH3, in Lilium longiflorum . Planta 197, 289–295 (1995). https://doi.org/10.1007/BF00202649
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DOI: https://doi.org/10.1007/BF00202649