[en] We have investigated the precise location of DNA within the meristematic cell nucleolus of Zea mays root cells and Pisum sativum cotyledonary buds, in the course of their activation and induced inactivation following a subsequent treatment at low temperature. For this purpose, we combined the acetylation method, providing an excellent distinction between the various nucleolar components, with the in situ terminal deoxynucleotidyl transferase-immunogold technique, a highly sensitive method for detecting DNA at the ultrastructural level. In addition to the presence of DNA in the condensed chromatin associated with the nucleolus, we demonstrated that a significant label was detected in the nucleolus of quiescent cells in both plant models. Evident labels were also found in the dense fibrillar component of actived nucleoli. Whereas in inactivated nucleoli no significant label was observed within the dense fibrillar component, an intense label was seen over the large heterogeneous fibrillar centres only during inactivation. The granular component was never significantly labelled. These results appear to indicate that the DNA present in the dense fibrillar component of activated nucleoli withdraws from this structure during its inactivation and becomes incorporated in the large fibrillar centres. These observations suggest that in plant cells inactivation of rRNA genes is clearly accompanied by changes in the conformation of ribosomal chromatin.
Benavente R., Rose K. M., Reimer G., Hügle-Dörr B., Scheer U. Inhibition of nucleolar reformation after microinjection of antibodies to RNA polymerase into mitotic cells. J. Cell Biol. 105:1987;1483-1491.
Chouinard L. A. Localization of intranucleolar DNA in root meristematic cells ofAllium cepa. J. Cell. Sci. 6:1970;73-85.
Chouinard L. A. An electron-microscope study of the intranucleolar chromatin during nucleogenesis on root meristematic cells ofAllium cepa. J. Cell. Sci. 19:1975;85-101.
Conconi A., Widmer R., Koller T., Sogo J. Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle. Cell. 57:1989;753-761.
Crèvecoeur M., Deltour R., Bronchart R. Effects of subminimal temperature on physiology and ultrastructure ofZea mays. Can. J. Bot. 61:1983;1117-1125.
de Barsy T., Deltour R., Bronchart R. Study of nucleolar vacuolation and RNA synthesis in embryonic root cells ofZea mays. J. Cell Sci. 16:1974;95-112.
Deltour R. Nuclear activation during early germination of the higher plant embryo. J. Cell Sci. 75:1985;43-83.
Deltour R., de Barsy T. Nucleolar activation and vacuolation in embryo radicle cells during early germination. J. Cell. Sci. 76:1985;67-83.
Deltour R., Gautier A., Fakan J. Ultrastructural cytochemistry of the nucleus inZea mays. J. Cell. Sci. 40:1979;43-62.
Deltour R., Mosen H., Bronchart R. Three-dimensional electron microscopy of the internal nucleolus-associated chromatin and of the nucleolar vacuoles during early germination ofSinapis alba. J. Cell Sci. 86:1986;53-71.
Deltour R., Motte P. The nucleolonema of plant and animal cells: A comparison. Biol. Cell. 68:1990;368-377.
Foe V. E., Wilkinson L. E., Laird C. D. Comparative organization of active transcription units inOncopeltus fasciatus. Cell. 9:1976;131-146.
Franke W., Scheer U., Spring H., Trendelenburg M., Zentgraf H. Organization of nucleolar chromatin. The Cell Nucleus. 1979;Academic Press, New York. p. 49-95.
Heliot L., Kaplan H., Lucas L., Klein C., Beorchia A., Doco-Fenzy M., Ménager M., Thiry M., O'Donohue M-F., Ploton D. Electron tomography of metaphase nucleolar organizer regions: Evidence for a twisted-loop organization. Mol. Biol. Cell. 8:1997;2199-2216.
Jennane A., Landre P., Nougarède A. Nucleolus activation in pea cotyledonary buds during 24 hours after decapitation of the main stem: Cytochemical studies. Protoplasma. 136:1987;29-36.
Jordan E. G. Interpreting nucleolar structure: where are the transcribing genes. J. Cell. Sci. 98:1991;437-442.
Lafontaine J. G., Lord A. An ultrastructural and radioautographic investigation of the nucleonematal component of plant interphase nucleoli. J. Cell. Sci. 12:1973;369-383.
Martin M., Moreno Diaz de la Espina S. Immunolocalization of DNA at nucleolar structural components in onion cells. Chromosoma. 98:1989;368-377.
Melcak I., Risueno M. C., Raska I. Ultrastructural nonisotopic mapping of nucleolar transcription sites in onion protoplats. J. Struct. Biol. 116:1996;253-263.
Motte P., Loppes R., Ménager M., Deltour R. Three-dimensional electron microscopy of ribosomal chromatin in two higher plants: A cytochemical, immunocytochemical, and in situ hybridization approach. J. Histochem. Cytochem. 68:1991;5-11.
Nougarède A. Infrastructure des axillaires cotylédonaires duPisum sativum. Crit. Rev. Acad. Sci. Sér. D. 284:1977;25-27.
Nougarède A., Landre P., Jennane A. Intranucleolar visualization of nucleic acids and acidic proteins in inhibited and reactivated pea cotyledonary buds. Protoplasma. 156:1990;183-191.
Nougarède A., Rembur J., Rondet P. Réactivation du bourgeon cotylédonaire du pois en réponse à la kinétine. Can. J. Bot. 59:1981;590-603.
Puvion-Dutilleul F., Mazan S., Nicoloso M., Pichard E., Bachellerie J-P., Puvion E. Alterations of nucleolar ultrastructure and ribosome biogenesis by actinomycin D. Implications for U3 SnRNP function. Eur. J. Cell Biol. 58:1992;149-162.
Raska I., Dundr M., Koberna K., Melcak I., Risueno M-C. Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? A critical appraisal. J. Struct. Biol. 114:1995;1-22.
Risueno M. C., Medina F. J. The nucleolar structure in plant cells. Revisiones sobre Biologia Celular. 7:1986;1-143.
Risueno M. C., Testillano P. S. Cytochemistry and immunocytochemistry of nucleolar chromatin in plants. Micron. 25:1994;331-360.
Scheer U. Changes of nucleosome frequency in nucleolar and non-nucleolar chromatin as a function of transcription: an electron microscopic study. Cell. 13:1978;535-549.
Scheer U., Benavente R. Functional and dynamic aspects of mammalian nucleolus. BioEssays. 12:1990;1, 14-21.
Schwarzacher H. G., Wachtler F. The nucleolus. Anat. Embryol. 188:1993;515-536.
Testillano P. S., Gonzales-Mellendi P., Ahmadian P., Risueno M. C. The methylation-acetylation method: an ultrastructural cytochemistry for nucleic acids compatible with immunogold studies. J. Struct. Biol. 114:1995;123-139.
Thiry M. Highly sensitive immunodetection of DNA on sections with exogenous terminal deoxynucleotidyl transferase and non-isotopic nucleotide analogues. J. Histochem. Cytochem. 40:1992a;411-419.
Thiry M. Ultrastructural detection of DNA within the nucleolus by sensitive molecular immunocytochemistry. Exp. Cell. Res. 200:1992b;135-144.
Thiry M. Nucleic acid compartmentalization within the cell nucleus by in situ transferase immunogold techniques. Microsc. Res. Tech. 31:1995;4-21.
Thiry M., Daneholt B. Evaluation of the sensitivity of the terminal deoxynucleotidyl transferase-immunogold technique on Balbiani ring genes. J. Histochem. Cytochem. 46:1998;1-7.
Thiry M., Goessens G. The nucleolus during the cell cycle. Champman, Hall. Molecular Biology Intelligence, Unit 1. 1996;1-114 R. G. Landes, New York.
Thiry M. J., Jamison M., Gilloteaux J., Summers J-L., Goessens G. Ultrastructural nucleolar alterations induced by an ametantrone/poly r(A-U) complex. Exp. Cell Res. 236:1997;275-284.
Thiry M., Puvion-Dutilleul F. Differential distribution of single-stranded DNA, double-stranded DNA and RNA in adenovirus-induced intranuclear regions of HeLa cells. J. Histochem. Cytochem. 43:1995;749-759.
Thompson W. F., Beven A. F., Wells B., Shaw J-P. Sites of rDNA transcription are widely dispersed through the nucleolus inPisum sativum. Plant J. 12:1997;571-581.
Van de Walle C., Bernier G., Deltour R., Bronchart R. Sequence of reactivation of ribonucleic acid synthesis during early germination of the maize embryo. Plant Physiol. 157:1976;632-639.
Wachtler F., Stahl A. The nucleolus: A structural and functional interpretation. Micron. 24:1993;473-505.
Wassef M., Burglen J., Bernhard W. A new method for visualization of preribosomal granules in the nucleolus after acetylation. Biol. Cell. 34:1979;153-158.
Weibel E. R. Stereological principles for morphometry in electron microscope cytology. Int. Rev. Cytol. 26:1969;235-302.
Yano H., Sato S. Cytochemical localization of intranucleolar DNA and RNA inAllium cepa. J. Electron Microsc. 44:1995;337-343.