Cytochemistry and immunocytochemistry of nucleolar chromatin in plants

Micron, Vol. 25, No. 4, pp. 331-360, 1994 Cmvrieht 0 1994 ElsevierScienceLtd All rights reserved

Pergamon

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Cytochemistry

and Immunocytochemistry Chromatin in Plants MARIA C. RISUERO*t

of Nucleolar

and PILAR S. TESTILLANO*$

*Laboratory of Nuclear Organization during Plant Development, Department of Plant Biology, Centro de Investigaciones Biol6gicas, CSZC, Vekizquez 144, E-28006 Madrid, Spain SDepartmento CC. Morfolbgicas y Cirugia, Facultad de Medicina, Universidad Alcal de Henares, Madrid, Spain

Abstract-This review attempts to document the most relevant data currently available on the in situ localization of nucleolar chromatin on plant cells. The data provided by the most powerful and recent in situtechniques, such as DNA specific ultrastructural staining, immunogold labelling, in situ molecular cytochemistry, in situ hybridization or confocal microscopy, are summarized and discussed in the light of the potential and limitations of each individual methodology. The presence of DNA in both fibrillar centres and regions of the dense fibrillar component is extensively documented. Data on the nucleolar distribution of other important macromolecules involved in ribosomal transcription are also shown and referred to with regard to the location of DNA. The comparison with the available data on the animal cell nucleolus points towards models of similar functional organization in both plant and animal nucleoli. Key words: Plant nucleolus, nucleolar chromatin, rDNA, fibrillar centres, dense fibrillar component, cryoprocessing, immunogold labelling, in situ molecular cytochemistry, NAMA-Ur, nucleic acid cytochemistry, cytochemistry plus immunogold.

CONTENTS I. Introduction .......................................................................................................................... II. Nucleolar components ................................................................................................................ III. Nucleolar localization of DNA:. ..................................................................................................... A. Specific ultrastructural DNA staining .............................................................................................. B. Immunogold labelling with anti-DNA antibodies .................................................................................. C. In situ DNA molecular immunoelectron microscopy .............................................................................. D. Immunolocalization of newly replicated DNA .................................................................................... E. In situ hybridization and confocal microscopy ...................................................................................... IV. Nucleolar distribution of other macromolecules with respect to DNA ................................................................ V. The metholdology for DNA in situ studies: Prospects and limits ..................................................................... VI. Functional organization of the nucleolus ............................................................................................. VII. Concluding remarks .................................................................................................................. Acknowledgements ................................................................................................................... References ............................................................................................................................

I. INTRODUCTION

It is well established that the nucleolus is the site where ribosomal RNA (rRNA) is transcribed, processed and assembled into preribosomal particles (Goessens, 1984; Hadjiolov, 1985; Scheer and Benavente, 1990). This prominent nuclear compartment constitutes the morphological expression of the rRNA genes whose activity dramatically affects the arrangement and organization of the different nucleolar components (Puvion and Moyne, 1981; HernHndez-Verdun, 1983, 1986, 1991; Fakan and tTo whom correspondence should be addressed. Abbreviations-bp, base pairs; BrdU, 5-bromo-deoxy-uridine; DFC, dense fibrillar component; DNase, deoxynuclease; FCs, fibrillar centres; GC, granular component; MA, methylation and acetylation; NAC, nucleolar associated chromatin; NOR, nucleolar organizer region; RNA pol I, RNA polymerase I; RNase, ribonuclease; RNP, ribonucleoprotein; TdT, terminal deoxy-nucleotidyl transferase; UBF, upstream binding factor.

331 333 .335 331 .337 .338 .344 344 .347 .348 .353 354 354 354

Hernindez-Verdun, 1986; Jordan, 1987, 1991; Risuefio and Medina, 1986; Thiry and Goessens, 1986; Schwarzacher and Watchler, 1991; Raska et al., 1992; Risuefio, 1993). This advantage, not available for many genes, makes the ribosomal transcription and the nucleolus excellent systems for studying transcription and control of gene expression. The nucleolus contains the rDNA and the different transcripts as well as all the structural and enzymatic molecules involved in such activity, i.e. the ribosomal proteins, RNA polymerase I, DNA topoisomerases, transcription factors, processing enzymes, the small nucleolar RNAs (U3, U8, U13 and U14) (Liihrmann et al., 1982; Lacoste-Royal and Simard, 1984; Parker and Steitz, 1987; Tyc and Steitz, 1989; Hughes and Ares, 1991; Girard et al., 1992; Li and Fournier, 1992) and also the 5s rRNA which is transcribed outside the nucleolus and imported into it, to be later assembled in the preribosomal particles (Sommerville, 1986; Reeder,

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1990; Warner, 1990; Sollner-Webb and Mongey, 1991). Other functions have recently been suggested for the nucleolus, e.g. a role in the transport and/or turnover of potential mRNAs (Ochs and Press, 1992; Bond and Wold, 1993). The genes specifying the rRNA are organized in tandemly repeated units which contain the regions encoding the 18S, 5.8s and 2% ribosomal RNAs, and an extended intergenic spacer (Walbot and Cullis, 1985; see reviews by Flavell, 1986; Flavell et al., 1986; Leitz and Heslop-Harrison, 1993). The rDNA units occur in tandem arrays at one or more chromosomal loci, the socalled nucleolar organizing regions (NORs) (McClintock, 1934; Godward, 1950; Givens and Phillips, 1986; Fedoroff and Botstein, 1992; Shapiro, 1992), first identified in mitotic chromosomes as the secondary constriction. The number of NORs per chromosome complement is usually small, e.g. one or two, though polyploids have rather more. The length of the rDNA repeat-unit varies between species (from 7000 to 12,000 bp in plants), but the gene regions mostly show substantial sequence homology not only between plant species (Appels et al., 1980; Flavell, 1986; Rogers and Bendich, 1987; Delseny et al., 1990) but also among eukaryotes (Appels and Honeycut, 1986). In contrast, the intergenic spacers show considerable sequence divergence between phylogenetic groups and, even, between plant species (Dvorak and Appels, 1982; Gustafson et al., 1988; Gruendel et al., 1991; Mukai et al., 1991; Cordesse et al., 1992; Tremousaygue et al., 1992); they are the sites for several elements involved in the regulation of the rRNA genes (Reeder, 1984, 1989; Grummt et al., 1985; Echeverria et al., 1992). The number of the rDNA repeat-units is much higher in plants than in other eukaryotes: from about 1200 to 31,000 in plants (Ingle, 1979; Flavell, 1986), while in mammalians the number is in the hundreds (Hadjiolov, 1985). In all cases the number of rRNA genes is greater than that required to sustain ribosome synthesis, even in growing cells (Macgregor et al., 1977; Buescher et al., 1984). This excess in the occurrence of rRNA genes raises many questions, especially in plants where the proportion of rDNA with respect to the total genome is very high. One of these questions is how such an enormous amount of rDNA is organized into the nucleolus. The chromatin spreading technique (Miller and Beatty, 1969; Miller, 1981), showing the well-known ‘Christmas tree-like’ images, have shown in a convincing way the extreme condensation that the ribosomal genes should have in an interphasic nucleolus (Greimers and Deltour, 198 1,1984; Trendelenburg, 1983; Scheer, 1987; Fakan and Hughes, 1989; Spring and Trendelenburg, 1990; Meissner et al., 1991). Nevertheless, this approach does not provide information about the intranucleolar localization of these genes. In the last few years, much progress has been made in identifying sequence elements of the ribosomal transcription unit in different organisms. Less is known about the chromatin structure of the ribosomal genes, especially in

plants. The use of psoralen crosslinking to rDNA in spreading techniques has allowed the distinction between active and inactive chromatin regions without degradation of the DNA in very different organisms of the phylogenetic scale (Sogo et al., 1984; Thoma and Sogo, 1988). It is based on the higher accessibility of active and open DNA to psoralen, due to the loss of histone-DNA interactions, and the relative protection of the inactive regions which are in a nucleosomal structure (Conconi et al., 1987; Lucchinni and Sogo, 1992; Dammann et al., 1993). Recently, psoralen crosslinking studies have shown two different structures in the ribosomal chromatin of a plant (tomato): ‘closed’ and ‘open’, the majority of the ribosomal genes (about 80%) being folded in a nucleosomal structure, similar to the bulk inactive chromatin (Conconi et al., 1992) (Fig. 1). Nevertheless, these results cannot answer the question as to whether the histones remain in the vicinity or are associated with DNA in another way to facilitate the rapid packaging of the genes after transcription. In fact, there is no consensus in the in situ studies over mammalian cell nucleoli reporting the presence or absence of histones in ribosomal chromatin (Hernandez-Verdun and Derenzini, 1983; Derenzini et al., 1983, 1985, 1987a, 198713, 1990, 1993; Thiry and Miiller, 1989; Raska et al., 1990,1992; Raska and Dundr, 1993). An important effort to relate in vitro observations to studies of gene activity in situ was the development of the mild loosening of the nuclear content (Puvion-Dutilleul and Puvion, 1980; Puvion-Dutilleul et al., 1981). However, the exact correlation between the structures revealed by this method and the nucleolar components has not been completely established. There have been numerous attempts to elucidate the ultrastructural localization of the nucleolar chromatin, mainly in animal cells, very often reporting contradictory data (Gosh and Paweletz, 1987, 1990; Thiry et al., 1988a, 1988b, 1993; Jimtnez-Garcia et al., 1989, 1992; Watchler et al., 1989, 1990, 1992, 1993; Raska et al., 1990, 1992; Puvion-Dutilleul et al., 1991; Stahl et al., 1991; Thiry and Thiry-Blaise, 1989, 1991; Thiry and Goessens, 1992; Derenzini et al., 1993; Hozak et al., 1993; Raska and Dundr, 1993; Scheer et al., 1993), but relatively few have dealt with plant cells (Deltour et al., 1979; Risuefio et al., 1982; Martin et al., 1989; Jordan and Rawlins, 1990; Rawlins and Shaw, 1990; Bertaux et al., 1991; Motte et al., 1991; Risueiio et al., 1991b; Testillano et al., 1991,1993b; Jordan et al., 1992; Leitch et al., 1992; Highett et al., 1993; Risuefio, 1993). Recently, the modern in situ cell biology methods including techniques such as immunogold labelling, in situ hybridization or confocal microscopy, are giving new insights to the understanding of the functional organization of the plant nucleolus. In this review, we attempt to document the most relevant data obtained to date on the in situ ultrastructural localization of nucleolar chromatin on plant cells. These results, the efficacy and problems of the methodology and the prospectives opened are also discussed in the light of the present understanding of the functional organization of the nucleolus.

Plant Nucleolar Chromatin in situ

II. NUCLEOLAR COMPONENTS In almost all nucleoli, different regions can be identified at the ultrastructural level: fibrillar centres (FCs), dense fibrillar component (DFC), granular component (GC) and nucleolar vacuoles (Yasuzumi and Sugihara, 1965; Goessens, 1984; Jordan, 1984; Cadrid and Lafontaine, 1985; Hernindez-Verdun, 1986; Hozak et al., 1986; Antoine et al., 1988; Crespo et al., 1988; Bedo and Webb, 1989; Lafarga et al., 1989, 1991; Derenzini et al., 1990; Scheer and Benavente, 1990; Derenzini and Ploton, 1991). The DFC is composed of 10 nm ribonucleoprotein (RNP) fibres tightly packed. FCs are small clear areas embedded in the DFC, containing fibrillar material in them. In plants two types of FCs have been described in relation to the transcriptional activity: homogeneous FCs (horn FCs) showing decondensed chromatin, which are

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small and numerous; heterogeneous FCs (het FCs) that contain decondensed chromatin fibres together with condensed chromatin inclusions of different sizes, are bigger and less numerous than the horn FCs. An intermediate type of FCs with structurally intermediate features between both types of FCs has also been detected (Risuefio et al., 1982, 1988b; Jordan et al., 1982; Medina et al., 1983a, 1983b; Risuefio and Medina, 1986). Nucleolar vacuoles are regions of lower electron density than the FCs. There are two types of vacuoles depending on the activity: reactivation vacuoles which are present in the FCs and are observed during the nucleolar reactivation processes, i.e. Gl cycling cells or early germinated plant embryos, and active vacuoles that are not present in the FCs. They contain RNP granules and are seen in G2. In highly active nucleoli a unique and large vacuole is often seen (Galan-Cano et al., 1975; Moreno-

conditions. (a) rDNA from Dicfyostelium discoidem. Fig. 1. Psoralen-crossfinked DNA, extracted and spread under denaturing -_ . -__. . . . . . Arrows indicate the ends of the heavily crosslinked rDNA regions wtuch do not show nucleosomal structure. The other rDNA remans show single-stranded bubbles corresponding to nucleosomes. Asterisks indicate the ends of rDNA palindrome. Arrowheads point out the plasmid pBR322, co-prepared as internal control of the spreading. Bar reoresents 1 urn. (b) Tomato leaf DNA. Small sinalestranded DNA bubbles represent mononucleosomes (small arrows) aid large bubbles corresp&I to dinucleosomes (arrow-heads). Large arrow points out a circular simian virus DNA, co-prepared as a control. Bar represents 1 kbp. (Courtesy of Dr J. M. Sogo, reproduced with permission from Sogo et al., 1984 and Conconi et al., 1992.)

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Fig. 2.

Plant Nucleolar Chromatin in situ

Diaz de la Espina et al., 1980; Fakan and HernandezVerdun, 1986; Risuefio and Medina, 1986; Risuefio et al., 1988b). The term interstice is frequently used to describe smaller clear areas between other nucleolar components in the animal nucleolus, and it has also been used for the plant nucleolus to define certain small vacuoles (Jordan, 1984). The shell of condensed chromatin which surrounds and penetrates into the animal cell nucleolus, the so-called nucleolar associated chromatin or NAC, is not present in the plant nucleolus. The only plant intranucleolar condensed chromatin is that of the het FCs (Risuefio et al., 1982; Risueiio and Medina, 1986; Testillano et al., 1991; Leitz et al., 1992; Highett et al., 1993). The nucleolus can be considered as a marker of cellular activity, because, changes in transcriptional activity are rapidly expressed by changes in its size, organization and structure (Miller and Knowland, 1970; Jordan and Chapman, 1971; Shermoen and Kiefer, 1975: Luck and Jordan, 1977; Risuefio et al., 1982; Hernandez-Verdun, 1983, 1991; Goessens, 1984; Sanchez-Pina et al., 1984; Martini and Flavell, 1985; Cabello et al., 1986; Risueiio and Medina, 1986; Olmedilla et al., 1987; Derenzini and Ploton, 1991; Puvion-Dutilleul et al., 1992). An active nucleolus shows an abundant granular component dispersed within the DFC, and which contains many horn FCs. A highly active nucleolus frequently shows large active vacuoles. However, a nucleolus with very low transcriptional activity is exclusively formed by DFC displaying large het FCs. When the nucleolus resumes activity the reactivation vacuoles appear and are connected with intermediate FCs (Risuefio et al., 1982, 1988b; Jordan et al., 1982; Medina et al., 1983a, 1983b; Risuefio and Medina, 1986). A rapid decrease in transcriptional activity produces a segregated morphology that in the plant nucleolus involves the GC surrounding the DFC. This typical morphology can be experimentally induced by cold and heat shocks, anti-metabolite treatments, RNApol I antibody microinjection (Benevente et al., 1988) etc., or can be found under various physiological conditions, as is the case of the vegetative cell of the pollen grain (Risueiio et al., 1972,1973; GalLn-Cano et al., 1975; Moreno-Diaz de la Espina and Risuefio, 1976; Medina and Risueiio, 1981; Medina et al., 1983a, 1983~; Olmedilla et al., 1987). Nucleolar components represent the various processes of rRNA synthesis, maturation, assembly and transport of preribosomal particles, and they clearly have varying molecular composition and structure. Ultrastructural

335

cytochemistry, therefore, allows a better visualization of the fibrillar and granular regions. A helpful method to distinguish these regions consists of the elimination or masking of some of their constituents without destroying the overall nucleolar organization (Wassef et al., 1979; Risueiio et al., 1982; Tandler and Solari, 1982; Risueiio and Medina, 1986; Thiry and Goessens, 1986; Biggiogera and Flach-Biggiogera, 1989; Derenzini and Farabegoli, 1990; Raska et al., 1990; Risuefio, 1993). The acetylation using acetic anhydride and pyridine, (Wassef, 1979; Wassef et al., 1979) has been used for visualization of RNP granules (Fakan and HernandezVerdun, 1986; Thiry and Goessens, 1986). A more efficient blocking agent for electron microscopy, the methanol-acetic anhydride (MA) (Tandler and Solari, 1982), provides a complete blocking of the stainable groups of proteins, the major constituents of the nucleolus, and, after uranyl and lead staining, enhances the distinction between granular and fibrillar elements (Fig. 2a,b). With this cytochemical method one can clearly define the limits of the FCs and DFC; on the other hand, no ultrastrucural evidence has been found for a ‘transition’ region between both fibrillar components, as has been postulated (Martin et al., 1989); neither has evidence of this ‘transition’ region been reported in the mammalian cell nucleolus (Hernandez-Verdun et al., 1991). The adaptation of the MA method to Lowicryl sections led to the application of new approaches such as the combination of this technique with immunocytochemical assays (Testillano et al., 1991; Gonzalez-Melendi et al., 1993; Risueiio, 1993; Mena et al., 1994). In addition, when EDTA regressive staining, preferential for RNPs, is performed on MA treated samples, the GC shows a higher contrast than the DFC, revealing the differential protein content of these components (Fig. 2~).

III. NUCLEOLAR

LOCALIZATION

OF DNA

Whilst many cytochemical techniques have been used to localize DNA in the nucleolus in epoxy or glycol metacrylate sections (Risuefio et al., 1982; Derezini et al., 1983, 1987b, 1990; Hernindez-Verdun, 1986; Risuefio and Medina, 1986; Biggiogera and Flach-Biggiogera, 1989; Motte et al., 1991), they have unequivocally detected it in the FCs, but fail to localize DNA in other nucleolar components. The difficulty in assuming that all rDNA is located in FCs has directed many investigations to develop new methodologies to solve the problem of the

Fig. 2. (opposite) Ultrastructural cytochemistry for nucleic acids in plant active nucleoli. Onion root meristematic cells, after formaldehyde fixation and Lowicryl embedding. (a) EDTA staining for RNPs, the nucleolus (NU) is preferentially stained, the different nucleolar components are not clearly distinguished, only the fibrillar centres (arrows) which appear as clear areas. (b) and (c) Methylation-acetylation (MA) method which blocks stainable groups of proteins. (b) Uranyl staining. The granular component (G) is clearly distinguished at the periphery of the nucleolus and intermingled with the dense fibrillar one (F). Two types of fibrillar centres are shown, the heterogeneous ones (arrowheads) which show condensed chromatin cores inside them, and the homogeneous ones (thick arrows) which are smaller and without condensed chromatin. Small vacuoles or interstices (v) can also be observed in relation to the granular component; they contain preribosomal granules. (c) EDTA staining after MA treatment. Due to its higher RNA content, the granular component (G) shows higher electron density than the dense fibrillar component (F). All types of fibrillar centres (arrows) appear bleached. Small vacuoles (v) containing EDTA stained granules can also be seen. Extranucleolar condensed chromatin masses (CHR) are bleached whereas fibrils and granules of the interchromatin regions (IR) are densely stained. Scale bars indicate 0.5 urn.

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Fig. 3. Osmium ammine B staining, specific for DNA. Regions of active nucleoli of Zea map, 120 hr after germination. 3 urn-Lowicryl sections and high pronase digestion. (a) Thin stained threads (arrowheads) interconnect two round, densely-stained areas (thin arrows) which correspond to fibrillar centres. (b) Stained fibres (arrowheads) link a fibrillar centre (thick arrow) and a stained peripheral knob of chromatin (k) which represents the NOR-chromatin that does not participate in the organization of the nucleolus. Other parts of the nucleolus (NU) appear completely bleached in both figures. Scale bars indicate 0.5 urn. (Courtesy of Dr R. Deltour.)

Plant Nucleolar Chromatin in situ

inaccessibility of rDNA fibrils to the majority of cytochemica1 procedures. A. Specific ultrastructural DNA staining For a long time the DFC was assumed to contain the nucleolar DNA, due to high resolution autoradiographical data after tritiated thymide or uridine incorporation which showed this fibrillar component to be labelled (Fig. 8c) (Granboulan and Granboulan, 1965; Smetana and Busch, 1974; Fakan, 1978, 1986; Mirre and Knibiehler, 1981; Puvion and Moyne, 1981; Risueiio et al., 1982; Goessens, 1984; Deltour and Mossen, 1987; Derenzini et al., 1990; Watchler et al., 1990). But, the inherent limitations of the technique that involves the comparatively large size of the silver grains together with the spreading of the radiation from its source, have led to the questioning of these results in different ways (Thiry and Goessens, 1991; Thiry et al., 1991). Indirect cytochemical methods such as DNase digestion has also indicated the presence of DNA in both types of FCs (Luck and Lafontaine, 1980; Risueiio et al., 1982; Derenzini et al., 1990). Ultrastructural cytochemical methods to specifically stain DNA are not abundant. The osmium ammine technique imparts good contrast to DNA without staining proteins (Cogliati and Gautier, 1973). Despite the fact that osmium ammine synthesis is a complex process, several groups have used it to study nucleolar chromatin (Deltour et al., 1979; Derenzini et al., 1982, 1993; Risueiio et al., 1982; Watchler et al., 1993). Condensed chromatin cores of the het FCs and fibres of the horn FCs are the only structures reported to be contrasted by this reagent (Risuefio et al., 1982; Risueiio and Medina, 1986). Recently, the use of a more stable and commercially available osmium ammine, the osmium ammine B, (Olins et al., 1989) on 3 pm thick sections after proteinase digestion has revealed densely-stained DNA fibres connecting those of different FCs (Fig. 3a) and FCs with the condensed NOR chromatin which does not participate in the organization of the nucleolus (Fig. 3b) (Motte et al., 1991). Within these nucleoli, such DNA connecting fibres should be localized at the DFC. The fact that the rest of the nucleolus appears extremely bleached presents some difficulty in the inequivocal identification of the round stained areas, the possibility that they include a stained layer of the DFC surrounding the FCs cannot be completely excluded. The new ultrastructural cytochemical method for DNA staining, the NAMA-Ur method, has been successfully applied to different plant and animal cells, and has also revealed DNA-containing structures in the nucleolus (Testillano et al., 1991; Derenzini et al., 1993; Risuefio et al., 1993). This simple and reproducible method uses ordinary chemicals and can be performed either en bloc or on Lowicryl ultrathin sections, and can be combined with immunogold detection (Fig. 6~). It also shows the nucleolus as a clear area where some condensed chromatin inclusions, corresponding to those of het FCs, appear

337

to be stained (Fig. 4a,b). Various stained fibres are also seen through the nucleolar body (Fig. 4c) or extending to the condensed chromatin cores (Fig. 4a,b). Occasionally, depending on the nucleolus, low electron density is induced within some nucleolar regions in the DFC by the NAMA-Ur method (Figs. 4 and 6~). B. Immunogold labelling with anti-DNA antibodies Presence of DNA in the FCs has been also reported from immunogold labelling using anti-DNA antibodies (Hansmann et al., 1986; Scheer et al., 1987; Thiry et al., 1988a,b, 1991; Martin et al., 1989; Raska et al., 1990; Motte et al., 1991; Risueiio et al., 1991b; Testillano et al., 1991; Moreno-Diaz de la Espina et al., 1992; Raska et al., 1992; Risueiio, 1993; Raska and Dundr, 1993). In some reports, a weak labelling was also noticed on certain regions of the DFC, using anti-DNA antibodies (Martin et al., 1989; Raska et al., 1990,1992; Martin and Medina, 1991; Risuefio et al., 1991b; Testillano et aE., 1991; Moreno-Diaz de la Espina et al., 1992; Gonzalez-Melendi et al., 1993; Raska and Dundr, 1993; Risuefio, 1993; Mena et al., 1994). These results should be interpreted carefully taking into account the fact that immunocytochemical methods provide good specificity and resolution, but they have the important limitation that only antigens exposed at the surface of the section can be detected (reviewed in Raska et al., 1990; Polak and Priestley, 1992; Merighi, 1992). In this way, ultrathin cryosections are a very convenient tool for immunoelectron microscopy because, in the absence of resin a greater number of epitopes are exposed to the antibodies. The superior antigenicity preservation provided by cryofixation methods constitutes an additional advantage to these approaches (Griffiths et al., 1984; Sanchez-Pina et al., 1990; Raska et al., 1990,1991b, 1992; Olmedilla et al., 1991, 1992; Risuefio et al., 1991a; Testillano et al., 1992a,b; Raska and Dundr, 1993). The ultrastructural cytochemical methods, when specific reveal macromolecules not only at the surface but also throughout the thickness of the section (Moyne, 1980). Only a few cytochemical techniques are able to combine with immunogold labelling, but this combination put together for the same section provides information concerning not only the presence of antigens but also the structures containing them (Risueiio et al., 1991a, 1993; Testillano et al., 1991, 1993a, 1993~; Thiry, 1988b, 1992~; Gonzalez-Melendi et al., 1993; Risuefio, 1993; Mena et al., 1994). The NAMA-Ur staining combined with anti-DNA immunogold labelling performed on Lowicryl sections reveal gold particles on the specifically stained structures (Risueiio et al., 1991b, 1993; Testillano et al., 1991,1993a, 1993~). By means of this combination, in the nucleolus, labelling appears on well-contrasted fibres and is dispersed over regions of the DFC showing a weak electron density (Fig. 6~). These anti-DNA labelled regions of the DFC are also observed in uranyl and lead stained Lowicryl sections (Fig. 5). When EDTA regressive staining is performed after anti-DNA labelling, the

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and P. S. Testillano

regions of the DFC that are labelled appear positively stained, indicating the presence of RNP material closely associated with DNA (Figs. 6a,b) (Risueiio et al., 1991b; GonzPlez-Melendi et al., 1994). These small EDTA positive regions of the DFC labelled by anti-DNA frequently appear near the FCs (Figs 5 and 6a,b). These data appear to support the hypothesis of Jordan (199 1) in which these regions are represented as narrow areas surrounding the FCs and that, depending on the plane section, they can be seen as isolated regions of the DFC. C. In situ DNA molecular immunoelectron

microscopy

New types of approaches have been recently developed to localize in situ DNA at different functional stages. They combine the accuracy and sensitivity of molecular biology reactions with the specificity and resolution of immunogold labelling methods. The in situ nick translation detects DNase I-sensitive sites of DNA, which to some extent might be related to their potential activity because the DNA sites which are active in transcription and/or replication, are in a more extended and accessible state for DNase I activity (Thiry, 1991a,b). The technique reproduces the nick translation reaction over ultrathin sections by incubating them with

DNase I, DNA polymerase I (DNA pol I), deoxinucleotides and biotinylated dUTP. The DNase I produces nicks in one of the DNA strands and the DNA pol I substitutes a fragment of native DNA with a new one which incorporates biotinilated dUTP; the biotinilated DNA is localized by an immunogold assay using antibiotin antibodies (Thiry, 1991a,b). The optimization of this technique for plant cryosections (Olmedilla et al., 1992) revealed new data on the study of the functional chromatin regions in plants. The results depend on the DNase I concentration used; high concentrations of the enzyme are needed to obtain labelling on the nucleolus (Fig. 7c) (Testillano et al., 1993b) confirming once more the unaccessibility of the nucleolar DNA. After 300 pg/ml DNase I, gold particles appear on the FCs and on some regions of the DFC close to the FCs. Under these conditions they also decorate extranucleolar condensed chromatin masses and dispersed DNA fibres at the interchromatin region (Fig. 7~). Another DNA molecular biology reaction adapted to ultrathin sections is that of the terminal deoxinucleotidyl transferase (TdT reaction). This highly sensitive enzyme incorporates biotin-labelled nucleotides to the free 3’hydroxyl ends of DNA, subsequent immunogold labelling with anti-biotin antibodies provides a very sensitive

Legends for pages 339 to 343 Fig. 4. NAMA-Ur staining specific for DNA on nuclei with different patterns of chromatin condensation. (a) and (b) Onion root meristematic cell nuclei, NAMA-Ur method performed en bloc. The chromatin containing structures are the only cellular components that are stained. The condensed chromatin masses (CHR) and fibres of dispersed chromatin in the interchromatin region (IR) appear contrasted; the extranucleolar chromatin appears less condensed in the Capsicum pollen (c) than in onion root meristematic cells (b). The nucleolus (NU) appears as a clear area showing the condensed chromatin cores (arrowheads) of the heterogeneous fibrillar centres to be well-contrasted. At high magnification (b), some stained fibres (arrows) can also be distinguished in the nucleolus. (c) Capsicum pollen grain nucleus, NAMA-Ur method performed on Lowicryl sections. The chromatin is more decondensed than in onion cells and appears as small stained patches connected by stained fibres of different thicknesses. The nucleolus (Nu) shows no contrast except for some thin fibres (arrows) which form a network. Scale bars indicate 0.5 pm. Fig. 5. (a) and (b) Immunogold labelling with anti-DNA antibodies and uranyl-lead staining. Nuclear regions of onion root meristematic cells, formaldehyde fixation and Lowicryl embedding. 10 nm-gold particles decorate the extranucleolar condensed chromatin masses (CHR). In the nucleolus (NU) labelling appears on the fibrillar centres (thick arrows) and over some regions (thin arrows) of the dense fibrillar component (F). G, granular component; IR, interchromatin region. Scale bars indicate 0.5 pm. Fig. 6. Anti-DNA immunolocalization combined with different cytochemical techniques. Nucleolar regions of onion root meristematic cells, formaldehyde fixation and Lowicryl embedding. (a) and (b) EDTA staining for ribonucleoproteins. Gold particles localizing DNA are seen (thin-arrows) in the dense fibiillar component (F) that are contrasted by EDTA and are frequently cl&e to the fibrillar centres, some of the latter also aDDear labelled (thick arrows). NU. nucleolus. (c) Anti-DNA localization and NAMA-Ur staining for DNA. Gold particles decorate t’he extranucleblar condensed chromatin patch;; (CHR) which are stained by the NAMAUr method. The nucleolus (NU) appears as a clear area with some regions showing a low electron density (asterisks); condensed chromatin of heterogeneous fibrillar centres (arrowheads) is also stained. Some anti-DNA labelling is seen in the nucleolus. Gold particles (thin arrows) appear near and over clearer areas, corresponding to the fibrillar centres (thick arrows). Neither labelling nor staining are observed in the cytoplasm (CT). Scale bars indicate 0.5 pm. Fig. 7. Ultrastructural localization ofDNA by the terminal deoxinucleotidyl transferase (TdT) reaction and in situ nick translation. (a) and (b) TdT reaction on Lowicryl sections, nucleolar regions of onion root meristematic cells. Gold particles decorate fibrillar centres (thick arrows) and extranucleolar chromatin (CHR). Labelling is also observed on the dense fibrillar component (F), in discrete regions (thin arrows) near the fibrillar centres. (c)In situ nick translation with high DNase I concentration. Ultrathin cryosection of a somatic cell of the Cap&urn anther. Labelling is seen on the fibrillar centre (thick arrow) and on certain regions (thin arrows) of the dense fibrillar component (F) near the fibrillar centre. Gold particles also appear over the extranucleolar condensed chromatin masses (CHR) and fibres (double arrows) in the perichromatin region. NU, nucleolus; G, granular component; IR, interchromatin region. Scale bars indicate 0.5 pm. Fig. 8. Ultrastructural localization of newly replicated DNA. (a) and (b) Bromo-deoxy-uridine (BrdU) incorporation during one third of the S phase and immunogold labelling with anti-BrdU antibodies. Ultrathin cryosections of onion root meristematic cells. (a) Nuclear region showing the labelling distributed over specific areas (arrows) of the reticulum formed by the condensed chromatin masses (CHR) and some fibres of the dispersed chromatin, while other chromatin areas appear unlabelled (asterisks). CT, cytoplasm; IR, interchromatin region. (b) Nucleolar region where gold particles are observed on certain regions (thin arrows) of the dense fibrillar component near the fibrillar centres (thick arrows). NU, nucleolus. (c) High resolution autoradiography after a long incorporation of tritiated thymidine. Silver grains are located over some fibrillar centres (thick arrows) and the dense fibrillar component (F). Extranucleolar condensed chromatin (CHR) is highly labelled. G, granular component. Scale bars indicate 0.5 pm.

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localization of DNA in situ, detecting very low quantities of DNA (Thiry, 1992a,b; Thiry et al., 1993). The use of this method on Lowicryl sections of plant cells has again clearly shown the presence of DNA on small regions of the DFC, near the FCs, as well as within the FCs (Figs. 7a,b) (Testillano et al., 1993b).

D. Immunolocalization of newly replicated DNA More information is provided by the use of anti-5bromodeoxi-uridine (anti-BrdU) technique. The in uivo incorporation of BrdU and subsequent ultrastructural detection of the incorporated precursors with anti-BrdU monoclonal antibodies, allows the study of DNA replication sites (De la Torre et al., 1985; Thiry and Dombrowicz, 1988; Thiry, 1988c, 1992b; Raska et al., 1990, 1991b; Humbert et al., 1992). Recently, this method has been successfully adapted for ultrathin cryosections of plant cells (Olmedilla et al., 1992). With a BrdU incorporation of approximately one third of the duration of the S phase (De la Torre and GonzLlez-Fernindez, 1979; De la Torre et aZ., 1985, 1991; Mergudich et al., 1992), nuclei show a different pattern of labelling depending on the cell cycle period of the cells (Fig. 8a). Concerning the nucleolus, when labelled, gold particles are seen on the FCs and/or regions of the DFC (Fig. 8b). This approach indicates that, independently of the analysis of the time and site of replication of rDNA, these nucleolar components contain newly replicated DNA. As explained above, the most recent approaches in the ultrastructural localization of DNA molecules are able to detect DNA not only in the FCs but also in the DFC, even though the whole area of this component seems not to react homogeneously with these techniques. The preferential labelling on small regions of DFC near the FCs could be explained by the difficulty of each individual method to reveal the whole nucleolar distribution of DNA and also by the existence of different functional areas in the DFC. These areas are more clearly distinguished in the large DFC of plant nucleolus than in other cells where this component is smaller, i.e. mammalian nucleoli (HernAndez-Verdun, 1986, 1991; Risuefio and Medina, 1986; Deltour and Motte, 1990; Derenzini et al., 1990).

E. In situ hybridization and confocal microscopy New insights are given by the in situ hybridization experiments; the localization of concrete genome sequences has been possible with this methodology (McFadden, 1989; Puvion-Dutilleul and Puvion, 1989, 1991; Puvion-Dutilleul et al., 1991; Olmedilla and Risueiio, 1993; Puvion-Dutilleul, 1993). The use of probes encompassing different fragments of the ribosomal transcription unit has been widely tested in recent years to localize either rDNA or its products (Sato and Shigematsu, 1985; Escaig-Haye et al., 1989; Jimtnez-Garcia et al., 1989; Thiry and Thiry-Blaise, 1989; Oakes and Lake,

1990; Oakes et al., 1990; Rawlins and Shaw, 1990; GCraud et al., 1991; Robert-Forte1 et al., 1991; Sato et al., 1991; Leitz et al., 1992; Highett et al., 1993). At the ultrastructural level ribosomal DNA/DNA in situ hybridization using the immunogold detection of digoxigenin or biotinlabelled probes has provided varying and controversial data on mammalian cells, by localizing the rDNA on the DFC (Gosh and Paweletz, 1990; Schwarzacher and Watchler, 1991; Watchler et al., 1989, 1990, 1992, 1993; Stahl et al., 1991; Hozak et al., 1993; JimCnez-Garcia et al., 1993), or at the interior and at the periphery of FCs (Thiry and Thiry-Blaise, 1989, 1991; Puvion-Dutilleul et al., 1991; Raska et al., 1992; Thiry and Goessens, 1992; Raska and Dundr, 1993). Recently, with a non-isotopic ultrastructural method, Dundr and Raska (1993) have localized rRNA transcription sites at the border of FCs. In plants there are few data available on ultrastructural rDNA localization from in situ hybridization. rDNA has been localized on the condensed chromatin bearing the NOR that appears in the nucleoli of quiescent maize embryos as two knobs adjacent to the periphery of the nucleolus (Motte et al., 1991). This rDNA located in the condensed chromatin masses forming the knobs could be the structural counterpart of the large amount of inactive rRNA genes detected by psoralen crosslinking in other plants (Conconi et al., 1992). This could also be the case for other cells where the high proportion of ribosomal chromatin is found on an inactive nucleosomal structure (Sogo et al., 1984) and could be mainly represented by the shell of condensed chromatin surrounding the nucleolus in many animal cells, the so-called nucleolar associated chromatin or NAC, where rDNA has been recently detected by electron microscopic in situ hybridization (Puvion-Dutilleul et al., 1991). On the other hand, even though it is generally accepted that nucleosomal structure is not compatible with transcriptional activity, there is still the question as to whether or not histones remain in the vicinity or are associated with active rDNA in another way to facilitate the rapid transition to the nucleosomal state. Different cytochetiical and immunogold labelling approaches have produced contradictory results on the presence of histones in nucleolar chromatin of mammalian cells (Derenzini et al., 1983, 1985, 1987a,b, 1990, 1993; Banchev et al., 1988; Thiry and Miiller, 1989; Raska et al., 1990, 1992; Raska and Dundr, 1993). Several attempts to immunolocalize histones H2B and H4 on plant cell nucleoli have shown labelling not only on the FCs but also at a low level on the DFC (GonzBlezMelendi et al., 1993). A different point of view is reached when whole cells are analyzed by confocal microscopy or wide field epifluorescence microscopy after in situ hybridization with fluorescent probes or specific DNA fluorescence dyes (for revision see Santisteban, 1993). The observation of optical sections using either wide field or confocal microscopy provides excellent three-dimensional images of subnuclear structures that are greatly improved by the application of data deconvolution because this removes the out-of-focus flare, providing much more image detail (Fig. 9) (Jordan and Rawlins, 1990; Rawlins and Shaw,

Plant Nucleolar

1990; Jordan et al., 1992; Highett et al., 1993; RobertForte1 et al., 1993). The DAPI (2,4 diaminophenylindole) is a very specific and sensitive fluorescent stain for DNA (Coleman et al., 1981; Otto and Tsou, 1985; Santisteban et al., 1992), that provides a useful way to study DNA distribution within the nucleolus, by using the above mentioned methodology (Montag et al., 1991; Spring et al., 1988; Jordan et al., 1992). It has been used in Spirogyra nucleolus, revealing small bright spots together with widely distributed bands and fibres, that are clearly seen after deconvolution (Fig. 9). Two bright knobs of condensed chromatin corresponding to the NORs of this species are also detected (Fig. 9). The study of these focal sections showed that DNA is more widely distributed than would be expected if FCs are indeed the only nucleolar component containing DNA (Jordan et al., 1992). Fluorescent labelled probes enable the use of optical tomography for in situ hybridization experiments. This allows the study of the three-dimensional distribution of rDNA through the nucleolus. Using fluorescent probes to rDNA, Highett and collaborators have recently determined the arrangement of ribosomal chromatin within the nucleolus of Pisum satioum root cells (Highett et al., 1993). In central focal sections of nucleoli, bright foci and some weakly labelled structures appeared to be connected in an extensive network (Fig. lOa); four bright ‘knobs’ corresponding to the NORs in this species, are also seen

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penetrating into the nucleolus (Fig. 10a). Changes in the distribution of the DNA are observed in relation to different transcriptional activity rates. One type of fluorescent pattern is observed in cells from the root cap, in which the internal chromatin is seen as larger foci indicating that the greater part of the rDNA is condensed within these nucleoli. Other nucleoli from the meristem show an extended decondensed fibrous network with some small bright foci (Fig. lob). These observations seem to be in agreement with the redistribution of nucleolar components at different stages of transcriptional activity. They show large het FCs in inactive or low active nucleoli from Allium cepa quiescent roots and Gl meristematic cycling cells. Active nucleoli in G2 meristematic cycling cells show small and numerous horn FCs and could be related to the images of rDNA distribution showing an extended chromatin network with small foci (Risueiio and Moreno-Diaz de la Espina, 1979; Risuefio et al., 1982,1988b; Medina et al., 1983a,b; Sato, 1985a,b; Risuefio and Medina, 1986). All this three-dimensional data strongly suggests that other nucleolar components, besides FCs, contain rDNA. On the other hand, confocal images clearly support the claim that plant nucleoli do not have the nucleolar associated chromatin (NAC) shell observed in mammalian cells. The individual ‘knobs’ located at the nucleolar periphery, which vary in number, corresponds to the NORs of the plant species. Therefore, the term

Fig. 9. Three consecutive focal sections (0.4 pm apart) of a Spirogyra grevelliana nucleus after DAPI staining. The upper images are unprocessed optical sections and the lower ones are the equivalent after deconvolution, which produces considerable improvement. The nucleolus corresponds to the darker inner circle. Numerous small bright spots, probably corresponding to the fibrillar centres are seen, as well as some bands and fibres widely distributed within the nucleolus. Scale bar represents 5 urn. (Courtesy of Dr P. Shaw and Dr E. G. Jordan, reproduced with permission from Jordan et al., 1992.)

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Fig. 10. In situ hybridization of rDNA on Pisum satiuum meristematic cell nucleoli with fluorescence probes, showing different fluorescence patterns related to their transcriptional activity rates. Stereo pairs from deconvoluted confocal data consisting of five optical sections 0.25 pm apart, sliced from the centre of the nucleoli. (a) The brightest four larger areas are the perinucleolar knobs (k). Fluorescence appears on small bright foci and many more faintly labelled structures that are interconnected in an extensive network. (b) More active nucleolus in which the internal rDNA is decondensed. Two bright knobs (k) are also observed. Scale bar represents 2 pm. (Courtesy of Dr P. Shaw, reproduced with permission from Highett et al., 1993.)

Plant Nucleolar Chromatin in situ

‘NAC’, suggested by Deltour et al. (Deltour, 1985; Deltour et al., 1986; Motte et al., 1988a, 1991), seems not to be adequate for the ‘knobs’ of the plant nucleoli.

IV. NUCLEOLAR DISTRIBUTION OF OTHER MACROMOLECULES WITH RESPECT TO DNA LOCALIZATION

The presence of proteins and/or RNA in the different nucleolar components and the relation of the DNA sites should be considered in order to understand the problem of accessibility of DNA to cytochemical techniques and the processes taking place in each nucleolar region. The different chemical compositions of the various nucleolar components, at least in terms of absolute concentrations, is supported by much cytochemical data (Yasuzumi and Sugihara, 1965; Deltour et al., 1979; Risuefio et al., 1982; Gas et al., 1984; Cadrin and Lafontaine, 1985; Escande-Geraud et al., 1985; Luck and Lafontaine, 1985; Moreno et al., 1985; HernandezVerdun, 1986; Risueiio and Medina, 1986; PuvionDutilleul and Laithier, 1987; Takeuchi, 1987; Antoine et al., 1988; Takeuchi and Takeuchi, 1988; Derenzini et al., 1990; Raska et al., 1990, 1992; Testillano et al., 1993~). Clearly, the proteins are the major constituent of the nucleolus. Many of them have been mapped by immunocytochemical approaches using poly- and monoclonal antibodies or autoantibodies from human diseases (Swanson-Beck, 1962; Kistler et al., 1984; SchmidtZachman et al., 1984; Escande et al., 1985; Gas et al., 1985; Hiigle et al., 1985; Lopez-Iglesias et al., 1988; Risuefio et al., 1988a; Pierron et al., 1989; Tan, 1989; Rasha et al., 1991a; Ochs and Press, 1992; Testillano, 1992b). Special mention should be considered for a set of nucleolar proteins which are able to reduce silver salts under acidic conditions, the so-called Ag-NOR proteins, which have been widely studied in many cellular types (Ploton et al., 1982,1983,1984,1985,1986,1987; Medina et al., 1983b, 1986; Sanchez-Pina et al., 1984; Moreno et al., 1985,1988,1990; Sato and Shigematsu, 1985; Risueiio and Medina, 1986; Thiry, 1986; Motte et al., 1988b; Risuefio et al., 1990; Pession et al., 1991). Their presence is necessary for ribosomal transcription, they have been located in the fibrillar components of the nucleolus, in association with rDNA (Medina et al., 1983b, 1986; Hernandez-Verdun, 1986, 1991; Risuefio and Medina, 1986; Derenzini and Ploton, 1991; Robert-Forte1 et al., 1993). At present, they have not been completely identified except for the major Ag-NOR proteins such as nucleolin (Gas et al., 1985; Roussel et al., 1992; Martin et al., 1992) and the B-23 (Biggiogera et al., 1989, 1991), leaving the identity of the rest of the Ag-NOR proteins still unknown. Since in the nucleolus, proteins are present in a much higher proportion than nucleic acids, they are probably masking the fine nucleic acid substructures. A useful approach for in situ detection in the nucleolus requires the removal of proteins by enzymatic digestion or preventing their contribution during staining. This later approach is

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very satisfactory because protein molecules remain in the ultrathin sections contributing to the maintenance of the nucleolar architecture. The methylation-acetylation (MA) method is one of the most efficient blocking techniques at the electron microscopic level (Tandler and Solari, 1982); it can be performed en bloc and on Lowicryl sections, and is easily combined with immunogold detections (Figs lla,b and 12) (Testillano et al., 1991; Gonzalez-Mendi et al., 1993; Mena et al., 1994). When MA-treated samples are stained with uranyl and lead, the distinction between the fibrillar and granular components of the nucleolus is greatly increased (Figs 2b, lla,b and 12), which is especially relevant in Lowicryl sections where such a distinction is often difficult (PuvionDutilleul et al., 1991) (compare Figs 2a and 2b). When combined with EDTA regressive staining, the MA method reveals the high protein content of the DFC in comparison to the other nucleolar components, the GC showing more electron density than the DFC (Fig. 2~). FCs and nucleolar vacuoles show a greatly different contrast and can be very well distinguished, due to the high electron density of particles inside the vacuoles (Fig. 2c). After DNA immunolocalization, gold particles appear on the FCs and small regions of the DFC which clearly shows a fine fibrillar ultrastructure (Fig. lla,b). These fibrillar regions are EDTA positive (Fig. 6a,b) revealing their RNP nature (Risueiio et al., 1991b; GonzalezMelendi et al., 1993). The enzymatic digestion with 20 pg/ml proteinase K on Lowicryl sections of MA-treated samples permits the loss of electron density of the DFC and reveals once more the high protein concentration of this component (Fig. llc,d). Regions showing a low but different electron opacity can be observed in the DFC after proteinase K digestion, the regions surrounding the FCs being more dense. Such dense regions appear irregularly distributed on the DFC and are interconnected forming a networklike association with the FCs (Fig. 1lc,d). The GC stands out from the bleached DFC. This constitutes an unusual image of the nucleolus in which the highly proteincontaining regions are distinguished by their low and differential opacity. If anti-DNA localization is performed after such proteinase K digestion, the gold particles are again found on FCs and regions of the DFC near the FCs (Fig. 1lc,d). When the simultaneous localization of DNA and RNA is performed on Lowicryl sections from MA-treated samples, using anti-DNA and anti-RNA antibodies (Eilat and Fischel, 1991), large particles corresponding to DNA are localized on FCs and small regions of the DFC. Small particles detecting RNA highly decorate the GC; a little labelling is also seen at the periphery of the FCs and in some regions of the DFC close to the FCs. This RNA localization agrees with data on animal nucleoli obtained using RNase-gold and immunogold methods (Raska et al., 1985; Thiry, 1988b, 1992c, 1993). This RNA labelling in fibrillar components occasionally appears near some large particles corresponding to DNA (Fig. 12) (Mena et al., 1994). Ultrastructural RNA/RNA in situ hybridiza-

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tion shows the presence of gold particles spread on the DFC and a little labelling in some FCs in the nucleolus of Ailium cepa root cycling cells (Olmedilla et al., 1993; Olmedilla and Risuefio, 1993). Even though the RNA polymerase I (RNA pol I) had first been immunolocalized in the FCs on mammalian cells (Scheer and Rose, 1984) and other reports had also found a prominent signal in FCs using anti-RNA pol I (Reimer et al., 1987a,b; Benavente et al., 1988; Rose et al., 1988; Raska et al., 1989, 1990), a few gold particles were also observed on the DFC of the HeLa cell nucleoli (Raska and Dundr, 1993) (Fig. 13a). When the upstream binding factor (UBF) associated to the RNA pol I (Voit et al., 1992) is immunolocalized in other animal cells, either exclusive labelling on the FCs and their periphery (Scheer et al., 1993; Zatsepina et al., 1993) or labelling on both the DFC and the FCs has been reported (Rendon et al., 1992; Rodrigo et al., 1992; Roussel et al., 1993). Only a little data on the in situ localization of the protein complex of ribosomal transcription is available in the plant nucleolus. Using the anti-NOR antibody, localizing the UBF, precise labelling on the DFC but not on the FCs has been reported on Lowicryl sections of the onion root meristematic cells (Rodrigo et al., 1992) (Fig. 13b,c). Other contradictory data using an anti-RNA polymerase II antibody crossreacting with a RNA polymerase I subunit have been reported, on LR White sections of the same material (Martin and Medina, 1991). The differences in the processing methods used could be one of the reasons for such differences in the results. Therefore, the exclusive localization of this important enzymatic complex cannot be stated yet. More recently, significant data have been provided by the use of anti-DNA/RNA hybrid antibodies at the ultrastructural level as a new method to map transcription sites in situ (Testillano et al., 1994). This approach clearly produced immunolabelled regions of the DFC and the periphery of some FCs in both mammalian and plant

Legendsfor

cells. In the latter case, in which the DFC is much larger, labelling of the DFC appears as small clusters, frequently near the FCs (Fig. 14). The presence of DNA in certain regions of the DFC, as documented in this review, and the immunolocalization of DNA/RNA hybrids in the same fibrillar regions are in agreement with the detection of the RNA polymerase I and the UBF in the DFC. All these data illustrate the possibility that the ribosomal transcription can take place in regions of the DFC near the FCs.

V. THE METHODOLOGY STUDIES: PROSPECTS

FOR DNA IA’ AND LIMITS

SITU

The supramolecular organization of the nucleolus is a very good model for studying advantages and limitations of molecular and modern in situ cell biology techniques. In the nucleolus, a high quantity of different macromolecules are organized in a very limited space producing dense regions where complex molecular processes are ordered and packed (Sommerville, 1986; Reeder, 1990; Warner, 1990; Fischer et al., 1991). The transcription and processing of ribosomal RNA are located in highly compact regions compared to extranucleolar transcription. This is probably one of the reasons why the cytochemical and immunocytochemical procedures have been less conclusive in analyzing nucleolar organization than other subnuclear compartments. Nevertheless, the in situ approach, which does not destroy the supramolecular organization, provides unique information on the in vivo organization of the process of ribosome biogenesis and can help to provide answers to within which subnucleolar structures the well-known spatially ordered molecular reactions are located. The information given by one individual immunocytochemical or in situ hybridization method, such as those described here, can perhaps seem limited but never trivial

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Fig. 11. Immunolocahzation of DNA combined with methylationacetylation (MA) treatment. Nuclear regions of onion root meristematic cells, formaldehyde fixation, Lowicryl embedding. (a) and (b) Uranyl and lead staining. Gold particles appear on the fibrillar centres (thick arrows) and on certain regions (thin arrows) of the dense fibrillar component (F), frequently near the fibrillar centres. High labelling is observed on the extranucleolar condensed chromatin patches (CHR). G, granular component. (c) and (d) Proteinase K digestion (20 ug/ml for 1 hr), the dense fibrillar component (F) shows low electron density due to the loss of its protein content, whereas the granular component (G) apears more highly contrasted; the fibrillar material and chromatin cores of the fibrillar centres (thick arrows) are also stained. The pattern of anti-DNA labelling is similar to that on non-digested sections: gold particles are observed on the fibrillar centres and regions (thin arrows) of the dense fibrillar component near the fibrillar centres. Scale bars indicate 0.5 urn. Fig. 12. (a) and (b) Double immunolocalization of DNA and RNA. Onion root meristematic cell nuclei, methylation-acetylation (MA) method and Lowicryl embedding. IS nm and 10 nm gold particles localize DNA and RNA, respectively. Large particles decorate the extranucleolar condensed chromatin (CHR) and the chromatin cores of the heterogeneous fibrillar centres (arrowheads). Small particles are concentrated on the ribosome-rich areas of the cytoplasm (CT) and on the nucleolar granular component (G), which is dispersed within the dense fibrillar component (F). Some RNA labelling also appears at the periphery of the fibrillar centres (thin arrows). Some dispersed labelling corresponding to both DNA and RNA is seen on the dense fibrillar component (F) where, occasionally gold particles of both sizes appear together (double arrows). Scale bars indicate 0.5 urn. Fig. 13. Immunolocahzation of RNA polymerase I and its associated transcription factor UBF with anti-RNA polymerase I and antiNOR serum, respectively. (a) anti-RNA pol I immunogold labelling on an ultrathin cryosection of HeLa cell, labelling is concentrated on the fibrillar centres (thick arrows), a few gold particles (thin arrows) are seen on the dense fibrillar component (F). N, nucleoplasm. (Courtesy of Dr I. Raska.) (b) and (c) anti-NOR immunolocalization on Lowicryl sections of AIIium cepa root meristematic cells. Gold particles are widely distributed over the dense fibrillar component (F) whereas all types of fibrillar centres (arrows) do not show labelling. CHR, extranucleolar condensed chromatin masses. Scale bars represent 1 pm. (Courtesy of Dr F. J. Moreno, reproduced with permission from Rodrigo et al., 1992.)

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and, considered together with many other in situ data, can add to the understanding of the functional organization of the nucleolus. In this way, the combination of the various ultrastructural techniques performed on the same section is to be strongly recommended. The fact that one labelled probe can hybridize with specific DNA fragments in a section (Puvion-Dutilleul and Puvion, 1989, 199 1; Puvion-Dutilleul, 1993) as well as the development of the in situ nick translation or the TdT reaction for ultrathin sections (Thiry, 1991b, 1992c), have opened new insights into the localization of specific DNA sites not only in animal but also in plant cells (Olmedilla et al., 1992,1993; Risueiio, 1993; Testillano et al., 1993b). In the post-embedding immunogold techniques the antibodies can only react with epitopes exposed at the surface of the section (Polak and Priestley, 1992). This limitation can be minimized with the use of ultrathin cryosection since in the absence of resin the epitopes are more accessible (Griffiths et al., 1984; Kellenberger et al., 1987; Sanchez-Pina et al., 1990; Raska et al., 1990; Olmedilla et al., 1991; Risuefio et al., 1991a; Testillano et al., 1992a,b). In contrast, cytochemical staining penetrates through the whole thickness of sections, providing electron density to specific structures. Thus, the simultaneous use of both cytochemical and immunogold

methods informs about the precise localization of an antigen and the structure supporting it (Risueiio, 1993). Such combinations of methods have been particularly useful for the nucleolar localization of DNA, as illustrated by the results presented. In all immunogold methods the determination of the optimum conditions for tissue processing and labelling of each sample and antibody is an essential prerequisite, especially when labelling with few gold particles is observed. Excellent results have been reported using low temperature processing methods including the ultrathin cryosections and Lowicryl sections (Bendayan et al., 1987; Kellenberger et al., 1987; Horowitz and Woodcock, 1992). The statistical evaluation of the labelling density over different subcellular components can be a help in understanding the results of an immunogold method which provides low labelling, but the numerical data obtained should be carefully evaluated. The conditions required to obtain reliable quantitative data include the performance of adequate controls with very low or null levels of background, as well as an even distribution of the gold particles (with no doublets or agglomerates) on clearly distinguishable subcellular components (see for revision Stirling, 1990; Merighi, 1992). In the last few years, the quantification of gold labelling density has been

Fig. 14. Immunogold labelling with anti-DNA/RNA hybrid antibodies. Nucleolar region of onion root meristematic cells, Lowicryl section. Gold particles are distributed either isolated or in small clusters on regions (thin arrows) of the dense fibrillar component (F), some of them being close to the fibrillar centres (thick arrows). Scale bar indicates 0.5 pm.

Plant Nucleolar

Chromatin

commonly reported in papers dealing with immunogold experiments; these numerical data are extremely useful for establishing comparisons, but they should not be taken as totally conclusive, especially when the prerequisites mentioned above are not observed; this could be the source of several of the contradictory observations on in situ localization of antigens which are scarce or masked, as in the case with those of nucleolar DNA. On the other hand, ultrastructural data should be carefully considered and interpreted in relation to the three-dimensional arrangement of the nucleolus. Confocal microscopy constitutes a very convenient approach that provides the three-dimensional information needed to understand ultrastructural data in a spatial perspective (Spring et al., 1988; Masson et al., 1990; HernandezVerdun et al., 1991b; Montag et al., 1991; Gautier et al., 1992; Humbert et al., 1992; Jordan et al., 1992; Santisteban et al., 1992; Santisteban, 1993). In situ hybridization techniques detecting ribosomal genes, when analyzed under confocal microscopy have offered the visualization of rDNA over a wider distribution than expected from the ultrastructural data (Highett et al., 1993; Robert-Forte1 et al., 1993). Moreover, this approach strongly suggests that DNA in the nucleolus is not restricted to the FCs, as postulated by several authors.

VI. FUNCTIONAL ORGANIZATION NUCLEOLUS

OF THE

Many of the most powerful and recent methods for localizing DNA in situ, such as immunogold labelling, in situ molecular cytochemistry, NAMA-Ur staining, BrdU incorporation and in situ hybridization, have shown the presence of DNA not only in the FCs but also in the DFC. In the latter, labelling was preferentially obtained in regions near the FCs. Many of the documented results illustrate the existence Table 1. Summary

of the differential

staining

353

in situ

of two different functional regions in the large DFC of the plant cell nucleolus: one near the FCs and another distant from the FCs (see Table 1 for a summary). Reports using the Ag-NOR techniques have already suggested this because of the different staining properties for the DFC surrounding the FCs (Medina et al., 1983b, 1986; Sanchez-Pina et al., 1984; Risueiio and Medina, 1986; Motte et al., 1988b). In this review, it has been shown that this fibrillar region also possesses a different electron density when various cytochemical methods for protein staining are performed (Figs 2c and llc,d), thereby illustrating the irregular distribution of the components forming the whole DFC. The main difference between the plant cell nucleolus and that of animal cells is the number of rRNA genes, which is much higher in plants (see Introduction). This could be one reason for the large size of the DFC in these nucleoli compared to the DFC of animal cell nucleoli where, typically, this component is restricted to a thin layer surrounding the FCs (Fig. 13a) (HernandezVerdun, 1986; Derenzini et al., 1990; Derenzini and Ploton, 1991; Schwarzacher and Watchler, 1991). The differential distribution of DNA and other macromolecules in regions of the DFC near to the FCs points to a new functional region in the large DFC associated with the FCs. Both fibrillar components (FCs and the DFC surrounding them) could be involved in transcription, even though the precise reactions taking place in each location cannot yet be determined. The regions of the DFC near the FC correspond to the striped areas shown in the diagram in Fig. 15. These could be analogous to those represented by Jordan (1991) in his scheme for the plant nucleolus. In this way, these fibrillar regions could be homologous to the DFC surrounding the FCs in the mammalian cell nucleolus. This interpretation could lead to an understanding of the nucleolar organization in a more general view, where the differences between plant and animal cell nucleoli are mainly

properties shown by the different nuclear compartments immunocytochemical methods are performed

when various ultrastructural

Nucleolus

In situ techniques

horn

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

+ + ++

osmium ammine (DNA) NAMA-Ur (DNA) anti-DNA (DNA) TdT reaction (DNA) BrdU incorporation (replicated DNA) In situ NT, high DNase I concentration (DNA) anti-RNA (RNA) MA + Ur Pb (nucleic acids) MA + EDTA (RNA) MA + Proteinase K (nucleic acids) anti-RNA pol I, mammalian (RNA pol I) anti-NOR (UBF)

FCs het cores

fP + -

++ ++ +++ +++ (+) ++ +++ _

f ++ -

++ -

I’+t +

near FCs ? + ++ ++ (+) + + ++ + xb ++

DFC far from FCs f f f _ + ++ ++ + -

cytochemical

and

Nucleoplasm

CC

Vacuoles

CHR

IR

_ _ _ _

_ _ _

++ ++ +++ +++ (++I

+ + + +

+++ ++ +++ ++

++ ++ + +

+++ +++ +++

(*) + +t ++ +++ +

++

The specificity or preferential staining of the techniques follows each method, in brackets. The symbols represent the intensity of gold labelling (in techniques 3%7,ll and 12) and comparative electron density (in techniques 1,2 and 8-10): + + +, very intense; + +, intense; +, normal; f , a few; -, absence. + p, indicates that labelling is preferentially seen at the periphery of the FCs. In technique 5, the brackets indicate that labelling only appears in certain nuclei which are synthesizing DNA. FCs, fibrillar centres; horn, homogeneous fibrillar centres; het cores, condensed chromatin cores of the heterogeneous fibrillar centres; DFC, dense fibrillar component; CC, granular component; CHR, condensed chromatin masses; IR, interchromatin region; NT, nick translation; MA, methylation-acetylation.

354

M. C. Risuefio and P. S. Testillano

quantitative. The existence of heterogeneous FCs, reported in plant nucleoli (Risuefio et al., 1982) could be interpreted as a condensed and inactive state of the excess of rDNA copies. A similar function could be assigned to the condensed chromatin shell of the mammalian cell nucleolus, where in situ hybridization experiments have localized rDNA (Puvion-Dutilleul et al., 1991), which does not appear in plant cells. More in situ studies are needed to confirm and establish such homologies. Nevertheless, the data presented here strongly support the functional organization of the plant DFC into two different regions which approach existing models of plant and animal cell nucleoli.

CONCLUDING

REMARKS

In summary, we have extensively documented the localization of DNA in the FCs and regions of the DFC near the FCs in the plant cell nucleolus, by revising data from the more powerful and recent in situ methodologies. All these methods are able to detect DNA in both the above mentioned fibrillar components. A comparison between the intranucleolar localization of DNA and other macromolecules has also been performed resulting in a distinction between two functional regions of the large DFC, i.e. close to and far from the FCs. All these data also suggest an involvement of the FCs and the surrounding regions of the DFC in transcription. The limitations and potential of the different in situ

techniques have been discussed in the knowledge that data from individual methods cannot answer the main questions concerning the exact localization of molecules and processes in the complex structure of the nucleolus. The determination of the best conditions for obtaining low background, precise localization of gold particles, combined with the performance of adequate controls, are essential prerequisites for the reliable quantification of labelling densities. Only by comparing the results from many different techniques and their integration, as illustrated in this review, can an understanding of the functional organization of the nucleolus be achieved. Moreover, a more general view of the nucleolar organization arises from the data presented here. A clear homology between the DFC in animal cell nucleolus and some regions of the DFC surrounding the FCs in plant cells is postulated. Acknowledgements-The authors wish to thank Prof. Dr Ivan Raska, and Drs Amelia Sgnchez-Pina, Adela Olmedilla, Miroslav Dundr, Concepcibn G. Mena and Pablo GonzBlez-Melendi for their collaboration in some of the experiments reported in this review; Dr Eduardo Gorab for his participation in the anti-DNA/RNA hybrid assays; Dr Eilat, Dr Fischell and Dr Stollar for their generous gift of the antibodies against RNA and DNA/RNA hybrids; Pablo GonzBlez-Melendi, Concepci6n G. Mena and Maria Angeles Ollacarizqueta for their help in the preparation of the review; Jo& Blanc0 for expert photographic work; Carlos Almarza for technical assistance; M. Victoria Lafita for her help in typing the manuscript and Beryl Ligus-Walker for checking the English style. This work was supported by grants from the Direcci6n General de Investigacidn Cientifica y TCcnica (DGICYT) PB87-0332CO2-01 and PB92-0079-C03-01.

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CONDENSED J4 a4

00

CHROMATIN

HOMOGENEOUS

FIBRILLAR

HETEROGENEOUS

FIBRILLAR

ii

DENSE

Q

GRANULAR

COMPONENT

0

NUCLEOLAR

VACUOLES

Fig. 15. Scheme

FIBRILLAR

CENTERS CENTERS

COMPONENT

of the nucleolar organization text, part VI).

in plant cells (see

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