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The P/ant Journal (1994) 6(6), 825-834 The maize RNA-binding protein, MA16, is a nucleolar protein located in the dense fibrillar component M. Mar AIb&, Francisco A. Culid~ez-Maci&, Adela Goday, Miguel Angel Freire, Bel6n Nadal and Montaerrat Pages* Departament de Gen~tica Molecular, Centre d'lnvestigacid i Desenvolupament C.S.I.C. Jorge Girona 18-26, 08034 Barcelona, Spain Summary A developmentally and environmentally regulated germ in maize, MA16, encoding an RNA-binding protein that binds preferentially to uridine and guanosinerich RNAs has previously been described. To gain some insight into the function of MA16 the distribution of MA16 mRNA and protein during maize development was investigated using in $itu hybridization, RNA and protein gel blot analysis and immunocytochemistry. The results show that MA16 is expressed throughout development of the embryo and seedling in different tissues and at different levels. The level of MA16 mRNA is higher in developing and expanding structures such as the root elongation zone and young leaves. After stress treatment MA16 mRNA increases in total and polysomal RNA, but no significant change in the level of the protein was detected. MA16 is a non-ribosomal nucleolar protein. Using immunoelectron microscopy the MA16 protein has been located in the dense fibrillar component and to a lesser extent in the granular component of the nucleolus. It was found that MA16 contains the conserved sequence motifs R(G)nY(G)eR and RR(E/D)(G)nY(G)n repeated in the Cterminal of the molecule that conforms imperfectly to the GAR motif proposed for nucleolar proteins. In light of these results the stress regulation of MA16 and a likely role for this protein in pre-rRNA processing and/or ribosome assembly is discussed. Introduction We have previously described a developmentally and environmentally regulated gene in maize MA16 which encodes a 16 kDa protein (Gomez et al., 1988). The protein sequence contains a single ribonucleoprotein consensus sequence type RNA-binding domain (RBD) in the amino terminus followed by a glycine-rich domain Received 30 March 1994; revised 20 June 1994; accepted 16 August 1994. *For correspondence (fax +34 3 2045904). in the carboxyl terminus. The predicted RNA-binding property of the MA16 protein synthesized in vitro was previously studied by using ribohomopolymer binding assays. Our results showed that the maize protein MA16 is an RNA-binding protein that binds preferentially to uridine and guanosine-rich RNAs (Ludevid et al., 1992). Regulation studies indicated that MA16 mRNA had a basal level of expression in several tissues including embryos and seedlings. We also showed that the level of mRNA in RNA gel blots increased after incubation with ABA or water stress treatment (Gbmez et al., 1988). Recently, the same effect has been observed in maize seedlings after ABA and iron stress treatments (Lobreaux et al., 1993). Genes homologous to MA16 have been identified in different plant species and specific regulation of their expression upon various environmental stresses such as wounding (Sturm, 1992), and chemical treatments (Didierjean et al., 1992) has been reported. Current evidence indicates that RNA molecules undergo diverse metabolic processes as a result of developmental or environmental cues, although the molecular mechanisms are largely unknown (for review see Dreyfuss et al., 1988). An increasing number of developmentally important RNPs have been identified in diverse organisms and have been implicated in a wide range of cellular processes including RNA processing, dbosome biogenesis and regulation of translation. The similar pattern of expression of MA16 gene homologues in response to different physiological conditions together with the highly conserved protein structure suggest a similar role for these proteins. As a first step in the identification of the function of the MA16 protein and to assess its importance in plant responses to environmental stress, we studied the distribution of MA16 mRNA and protein in maize tissues by using in situ hybridization, subcellular fractionation, and immunomicroscopy. Our results demonstrate that MA16 is widely distributed in different maize tissues and it is localized within the nucleolus and is thus the first member of the plant RNA-binding protein family which has been localized in the dense fibrillar component of the nucleolus. ResuRs Expression of MA 16 in embryos and seedlings To determine the expression and localization of MA16 protein different maize tissues were analysed by protein gel blot analysis and immunocytochemistry with anti825 826 M. MarAIb& et al. bodies raised against maize MA16 (Ludevid et al., 1992). We previously reported that the expression of MA16 mRNA increases in response to environmental stress in the plant, therefore we also examined whether there was any accumulation of the MA16 protein during water stress. Total proteins were extracted from embryos at 10, 20, 30, 40, 50, and 60 days after pollination (d.a.p.), and from well-watered or water-stressed seedlings 8 h after germination and 2 and 6 days old. Figure l(a) shows that substantial levels of MA16 were present in embryos at different developmental stages. A slightly higher level of the protein was detected at 30-40 d.a.p, when embryo desiccation starts. The MA16 protein was still present in mature seeds (50 d.a.p.) but it was barely detectable in dry embryos (60 d.a.p.) and in embryos after 8 h of germination. In general, protein levels during embryogenesis and early germination paralleled that of MA16 mRNA accumulation previously reported. MA16 mRNA was present at basal levels in immature embryos, the level of mRNA was higher in 40 d.a.p, embryos and it was barely detectable dudng the first hours of germination (Gbmez et al., 1988). During seed germination the level of the protein rises and then at about 2 days after germination it attains a basal level of expression. Figure l(b) shows that similar levels of the protein were detected in roots and leaves of 2- and 6-day-old plants. There is a small but constant difference of MA16 level between the leaves and the roots, the latter having a lower level of the protein. No significant change in the levels of the protein were observed in tissues under water stress. Since during water stress MA16 mRNA was significantly increased (Gbmez et al., 1988) but the amount of protein detected by Western blot analysis remained approximately the same, we wanted to rule out the possibility that MA16 mRNA was controlled at the translational level during stress treatment. To test this hypothesis the distribution of MA16 mRNA was analysed in total and polysomal RNA fractions from normal and water-stressed tissues. Polysomes were prepared from dry embryos and leaves from either wellwatered or water-stressed plants. Comparison of cytosolic and polysomal RNAs by Northern blot shows that there is a parallel increase in total and polysomal MA16 mRNA after stress treatments (Figure lc). It has been reported that a substantial amount of alternatively spliced messages of the RNA-binding protein RGP-1, which is the tobacco homologue of MA16, are localized in the polysomal fraction of different tissues (Hirose et al., 1993). These altematively spliced RGP-1 mRNAs were suggested to produce truncated polypeptides. In the case of MA16, alternatively spliced forms of the mRNA were not detected and a single transcript was found in either the cytoplasmic and the polysomal RNA fractions (Figure lc). Spatial distribution of MA 16 mRNA in maize seedlings Figure 1. Expressionof MA16in embryosand seedlings. (a) Immunodetectionof the MA16 protein by Western blot. Protein extractsfrom embryosof 10, 20, 30, 40, 50 and 60 d.a.p.(DAP)wereused. (b) Westernblotof seedlings:8 h (8H);2 days(2D),and 6 days(6D) after germination. Well-watered root (RC) and leaves (FC); root (RS) and leaves(FS) underwaterstress. (c) Northern blot of total and polysomalRNA, hybridizedwith a probe againstthe MA16mRNA. Leavesfrom 6-day-oldseedlingswithouttreatment (C) and after3 h ($1), 1 day ($2) and 3 days($3) of waterstress. Embryos 60 d.a.p. (E). Below, ethidium bromide staining of the correspondingRNA is shown. To further investigate the pattern of MA16 gene expression we localized MA16 mRNA and protein within the cells of various maize organs at different stages of development using in situ hybridization and immunocytochemistry. Figure 2 shows the distribution of MA16 mRNA and protein in seedlings. For in situ hybridization fixed sections were hybridized with digoxigenin-labelled MA16 antisense- or sense-strand probes (Figure 2a-f). Figure 2(a) and (c) show longitudinal sections of plumule and Nucleolar localization of the maize MA 16 protein 827 Figure 2. Localization of MA16mRNA and protein in maize seedlings. (a-f) In situ hybridization of longitudinal and transversal sections hybridized with digoxigenin-labelled antisense MA16probes and viewed under bright field which gives a purple label. Epifluorescence was used to reveal calcofluor-stained cell walls (bright). Longitudinal sections from the plumule in (a) and the radicle in (c) of 4-day-old seedlings and a transverse section from plumule in (b) are shown. More detailed views of (a) and (b) are shown in (d) and (e), respectively. A control hybridized with an MA16sense probe is shown in (f). (g-I) Immunolocalization of MA16 protein on paraffin-embedded sections incubated with anti-MA16 antibodies and an avidin-biotin-peroxidase detection system. Brown staining indicates an MA16 antibody-specific reaction. Longitudinal sections from the plumule in (g) and a transversal section from 7-day-old leaves in (h) are shown. A more detailed view of (h) is shown in (i). Control reactions using pre-immune serum are shown in (j), (k) and (I). Abbreviations: PL, plumule; C, coleoptile; LR, lateral root; EZ, elongation zone; RM, root meristem; RC, root cap; AM, apical meristem; VB, vascular bundle; E, epidermis; M, mesophyl; N, nucleus. Magnification x50 in (a--c), (g) and (j); xl00 in (d-f), and (h) and (k); x600 in (I) and (k). radicle and Figure 2(b) s h o w s transversal sections through the plumule from 4-day-old seedlings. The MA16 anti-sense p r o b e p r o d u c e d intense hybridization staining in the plumule leaves a n d lateral roots (Figure 2a a n d b). Hybridization staining w a s not detected, or w a s very low, in the apical m e r i s t e m a n d in the coleoptile (Figure 2d a n d e). In y o u n g roots, a c c u m u l a t i o n of MA16 m R N A w a s o b s e r v e d in d e v e l o p i n g cells (Figure 2c). In situ hybridization with a MA16 m R N A control p r o b e w a s used to m o n i t o r b a c k g r o u n d hybridization. T h e specificity of the reaction w a s s h o w n by the lack of a p p r e c i a b l e reaction of the MA16 sense-strand p r o b e with the p a r a f f i n - e m b e d d e d sections (Figure 2f). For i m m u n o c y t o c h e m i s t r y paraffin sections from different o r g a n s w e r e incubated with anti-MA16 antibodies (Figure 2g-I). Figure 2(g) and (h) s h o w that the MA16 828 M. Mar AIb~ et al. protein was evenly distributed in the different tissues and cell types. It was, however, enhanced in leaf (Figure 2h) in agreement with the in situ hybridization data. Figure 2(i) shows that anti-MA16 antibodies reacted very strongly with the nucleus, while no staining was observed with pre-immune serum (Figure 2j-I). The MA 16 is a non-ribosomal nucleolar protein To determine the intracellular location of the MA16 protein more precisely, immunodetection of the protein in subcellular fractions of maize tissues was performed. Nuclei were obtained from 30 d.a.p, embryos. Figure 3 shows electrophoretic profiles of embryo subcellular fractions (Figure 3a) and the distribution of the MA16 protein and histones H2A and H2B in each fraction analysed on protein gel blots with anti-MA16 and anti H2A-H2B antibodies (Figure 3b). MA16 was present in both cytosolic and nuclear fractions, whereas histones H2A-H2B were absent in the cytosolic fraction. Pudfied nuclei were sonicated and centrifuged on a cushion of sucrose as described in Experimental procedures. This procedure was reported to yield fractions either enriched in nucleoli (in the pellet) or in broken chromatin and ribonucleoprotein particles (on top of the sucrose cushion). After sedimentation of the sonicated nuclei three fractions were harvested: the pellet; the interface (the top of the sucrose cushion); and the supernatant. Protein gel blot analysis indicates that significant amounts of the MA16 protein are present in the nucleolar pellet (Figure 3c, lane 2) whereas the level of MA16 is reduced in the broken chromatin (interface fraction) (Figure 3c, lane 3). The supernatant fraction (containing soluble proteins) has a low level of the MA16 protein and histories H2A and H2B are absent in this fraction. A similar distribution of the MA16 protein was found in subcellular fractions of leaves of 6-day-old seedlings (results not shown). The possibility that MA16, which is a small protein of 16 kDa and pl 6, could be a ribosomal protein partially accumulated in the nucleolus was also investigated. Gel blot analysis of polydbosome proteins from dry embryos, and leaves well watered or after water-stress treatment (Figure 3d), indicates that MA16 is exclusively associated with the fraction free of polyribosomes (Figure 3e, lanes 5-8) and is not a component of the mature cytosolic ribosomes (Figure 3e, lanes 1-4). The MA 16 protein is located in the dense fibrillar component of the nucleolus To further confirm the subnuclear location of MA16, immunoelectron microscopy was performed. Thin sections of LR white-embedded tissues of maize were incubated Figure 3. Distribution of MA16 in subceltular fractions. (a) Coomassie blue-staining of total (T), cytoplasmic (C) and nuclear (N) protein fractions of 30 d.a.p, embryos. (b) Protein gel blot analysis of the fractions: in (a) using polyclonal antibodies against MA16 and against H2A/H2B. (c) Protein gel blot analysis of subnuclear fractions: 1, total nuclear fraction; 2, pellet (nucleolar-enriched fraction); 3, interface (chromatinenriched fraction); 4, supematant (soluble proteins fraction). (d) Coomassie blue-staining of the polysomal (POL) and frae-of-polysomes (SOL) protein fractions. Leaves from 6-day-old seedlings were used: without treatment (lanes 1 and 5), and after 3 h (lanes 2 and 6) and 1 day (lanes 3 and 7) of water-stress treatment. Embryos 60 d.a.p. (lanes 4 and 8). (e) Immunodetection of MA16 in the fractions shown in (d). Nucleolar localization of the maize MA 16 protein with anti-MA16 antibodies and subsequently detected by immunogold labelling. Figures 4 and 5 show electronmicrographs of maize sections of embryos at 30 and 50 d.a.p. (Figure 4) and leaves of 6-day-old seedlings (Figure 5). In all the cells studied the distribution of the gold particles in the different cellular compartments was similar. The MA16 protein was mainly detected in the nucleolus with some labelling also scattered throughout the nucleoplasm (Figures 4d and 5). Nucleoli from the cell types studied have a different ultrastructural organization. They show the three basic nucleolar components, the fibrillar centres (FC), the dense fibrillar component (DFC) and the granular component (GC) (Jordan, 1991; Scheer and Benavente, 1990). Several nucleoli showed nucleolar vacuoles of different size (Figures 4c and 5a). Some cells had compact nucleoli (Figure 5a), these nucleoli are exclusively fibrillar and have been described in cells under conditions of limited or halted transcriptional activity (RisueSo and Medina, 1986). Specific labelling was always observed in the dense fibdllar component and a lower signal was also seen in the periphery of the nucleolus where the granular component is localized (Figure 4b). In contrast, the fibrillar centres were devoid of labelling. When the nucleolus showed nucleolar vacuoles (Figure 4c) they appeared free of gold particles. Gold particles were also homogeneously distributed throughout the nucleoplasm (Figures 4d, 5a and b) in agreement with the nuclear staining observed with the optical microscope. The cytoplasm and cellular components such as cell wall, chloroplasts and organelles were devoid of labelling (Figure 4e). In control sections incubated with the pre-immune serum no specific labelling was observed in the nucleolus or in the nucleoplasm (Figure 4f). Sequence similarities between MA 16 and nucleolar proteins The MA16 protein contains two types of polypeptide domains (see Figure 6a), one with the conserved RNAbinding domain (Dreyfuss et al., 1988) and another with a glycine-rich domain interspersed with aromatic and charged amino acid residues (Gomez et aL, 1988). A distinct glycine/arginine-rich conserved motif (GAR domain) has been identified in several nucleolar proteins. It has been proposed that the GAR domain is restricted to nucleolar proteins (Girard et aL, 1992). This GAR domain is formed by the internal repetition of short stretches of five to 12 residues containing the sequence RGGXGGR or RGGXRGG where X is phenylalanine, serine, tyrosine or alanine. In the maize MA16 protein similar GAR-like motifs, R(G)nY(G)nR and RR(E/D)(G)nY(G)n, containing different numbers of G residues are repeated in the Cterminus half of the molecule. Other nuclear proteins with 829 glycine-rich regions have also been reported such as hnRNP A1 and A2 (Burd et al., 1989; Cobianchi et al., 1986). However, the amino acid composition of the glycine-rich domain is different from the GAR. In particular there is a large under-representation of arginine in A1 and A2 (6-8%) and there are several non-GRF residues scattered in the sequence. In an attempt to identify possible functions of MA16 we analysed the relationships of proteins containing the two interactive polypeptide domains, the RBD and the GARlike domain, using the multiple sequence alignment (Feng et al., 1987). Most of the proteins used in the comparison contain multiple RBDs. The RNA-binding domain included in the comparison is the one immediately upstream of the glycine-rich domain. The dendrogrem (Figure 6b) shows three groups of proteins which are likely to be functionally distinct classes. These classes are nucleolins, plant proteins and hnRNPs. The fact that all the plant proteins cluster together in the tree indicates that its primary sequence was highly conserved during evolution and suggests that they are functionally related. Interestingly, the topology of the tree also indicates a higher proximity of MA16 with the cluster of nucleolar proteins than with the nuclear hnRNP cluster. Discussion To investigate the possible function of MA16 we have used protein gel blot analysis and immunolocalization to characterize the pattern of accumulation of the MA16 protein in relation to plant development and desiccation conditions. The wide distribution of MA16 mRNA and protein found in this study suggests that it functions in many cells. MA16 is expressed throughout development of the embryo and seedling in different cell types and at different levels. In situ hybridization studies showed that MA16 mRNA is not uniformly distributed, it is present in vascular tissues and it accumulates in elongating and expanding structures such as the root elongation zone and young leaves. The MA16 accumulation and localization pattern suggest that it is probably involved in general growth phenomena in the plant. Interestingly, growth is inhibited by water deficit and in the elongating cells a decrease in the growth rate has been correlated with an increase in the ABA content (Bensen et aL, 1988). Under water deficit an increase in MA16 mRNA was obtained by using as a probe the 3' non-coding region of the MA16 cDNA, which by Southern analysis results in a single hybridizing band (Gomez, unpublished results). The level of MA16 mRNA has been shown to increase both in total RNA or polysomal RNA during water stress. However, no significant changes in the levels of the protein were observed in this study. Together, these results suggest that the accumulation 830 M. Mar AIb~ et al. Nucleolar localization of the maize MA 16 protein of MA16 mRNA observed under several environmental stresses may reflect specific alterations in development and growth of the cells (such as a decrease in their metabolism) rather than a direct response of the gene to desiccation. Immunoelectronmicroscopy showed accumulation of MA16 in the nucleolus. Nucleoli from the cell types studied have different morphologies depending on the tissue and the state of transcriptional activity. In all these nucleoli the immunogold labelling was found mainly in the DFC, and some labelling was also observed in the GC. The constant distribution of MA16 to specific structures within the different nucleoli suggests again a housekeeping role for the protein. Comparison of polyribosomal and nucleolar proteins by Western blotting using antiMA16 antibodies shows that MA16 is located in the nucleolus but is not part of mature cytoplasmic ribosomes. A set of non-ribosomal nucleolar proteins such as 831 nucleolin and other major nucleolar proteins have been shown to shuttle between nucleus and cytoplasm (Borer et al., 1989) and they have been involved in different steps of the biogenesis and transport of ribosomes. Moreover, several nucleolar proteins have been assigned to the various nucleolar components (reviewed by Nigg, 1988). Of these the B-36 protein (fibdllarin) has been identified in the DFC of plant cell nucleoli (Testillano et al., 1992). It has been suggested that the DFC is the major site of pre-RNA processing and preribosome assembly, while the GC probably contains ribosomal subunits awaiting transport to the cytoplasm (Tollervey et al., 1991). However, the explanation in molecular terms of the nucleolar components is still unclear (Jordan, 1991). The MA16 protein belongs to a small family of proteins that contain two types of interactive surfaces, one with the conserved RNA-binding sequence that has the Figure 5. Immunogold detection of MA16 on ultrathin sections of leaves. (a and b) Immunolocalization of MA16 protein on ultrathin sections of leaves of 6-day-old plants incubated with anti-MA16 antibodies and detected with protein A coupled with 10 nm gold particles. A compact nucleolus is shown in (a). Abbreviations: N, nucleoplasm; NU, nucleolus; V, vacuole. The bar represents 0.2 Hm. Figure 4. Immunogold detection of MA16 on ultrathin sections of embryos. Immunolocalization of MA16 protein on ultrathin sections of embryos incubated with anti-MA16 antibodies and detected with protein A coupled with 10 nm gold particles. (a) Nucleus (N) from a cell of the embryonic axis of a 30 d.a.p, embryo. (b) Nucleolus (NU) from a cell of the embryonic axis of a 30 d.a.p, embryo. (c) Nucleolus from a cell of the scutellum of a 30 d.a.p, embryo. (d) Detail of nucleoplasm (N) and nucleolus (NU) of a cell of the embryonic axis of a 50 d.a.p, embryo. (e) Detail of cytoplasm (C) and cell wall (CW) of the cell shown in (d). (f) Nucleolus from a cell of the embryonic axis of a 30 d.a.p, embryo incubated with pre-immune serum. The bar represents 0.2 I~m. Abbreviations: DFC, dense fibrillar component; GC, granular component; FC, fibdllar centre. V; vacuole. 832 M. M a r AIb& et al. (o) RBD 1 RNP2 88 GAR-LIKE 157 RNP1 R(G)nY(G)nR RR(D/E) (G)nY(G)n (b) i Hrp36dr Aldr A1h A2h _ _ ~ Figure 6. Schematicrepresentationof MA16protein and the dendrogram of MA16and proteinscontainingRBD and GAR-likedomains. (a) Structureof the MA16 protein. RBD, RNA-bindingdomain; GAR-LIKE, Gly-Arg-rich domain homologue. RNP1, RNP2: ribonucleoproteinconsensus sequences 1 and 2. The hatched boxes in the GAR-LIKE section represent repeatedsequences. (b) Dendrogram of MA16 and proteinscontainingRBD and GAR domains using the progressive alignment method of Feng et al. (1987). Nucm, mouse nucleolin(Bourbonand Amalric, 1990). Nucr, rat nucleolin(Bour10onand Amalric, 1990). Nucha; hamster nucleolin(Lapeyre et al., 1987). Nucch, chicken nucleolin(Maridorand Nigg, 1990). Nucx,Xenopus laevis nucleolin (Rankin et al., 1993). Grpls and Grp2s, Sorghum vulgare Glyrich proteins 1 and 2 (Cretin and Puigdomenech,1990). Chem2m, maize mercuric chloride-inducedprotein (Didierjean et aL, 1992). Ma16m, maize MA16 protein (Gbmez et al., 1988). Grpc, carrot Gly-rich protein (Sturm, 1992). Grpr, rape Gly-richprotein (Bergeron et al., 1993; EMBL Z14143). Grplan, Gly-rich protein la Nicotiana sylvestris (Hirose et aL, 1993). Grpa, Gly-rich protein Arabidopsis thaliana (van Nocker and Vierstra, 1993). Hrp36dr, heterogeneous nuclear RNP protein 36 Drosophila melanogaster (Matunis et al., 1992). Aldr, heterogeneous ribonuclear particle protein A1 homologue D. melanogaster (Haynes et al., 1990). Roaax, heterogeneousribonucleoproteinA1-A X. laevis (Kay et al., 1990). Alh, human hnRNP protein A1 (Cobianchi et al., 1986). A,?.h,human hnRNP protein A2 (Burd et al., 1989). Hrp40dr, hnRNP protein 40 D. melanogaster(Matunis eta/., 1992).Abh, human-typeA/B hnRNPprotein (Khan eta/., 1991). Roaax Abh ~ Nucm Nucr Nucha Nucch Nucx I -ii Grpls Chem2m Ma16m Grp2s Grpc Grplan Grpr Grpa Hrp40dr potential to bind RNA and another with a glycine-rich domain interspersed with aromatic and charged amino acid residues that may interact with other molecules. Interestingly, among the few proteins of the eukaryotic nucleolus that have been characterized, nucleolin (Lapeyre e t aL, 1987), fibrillarin (Ochs e t al., 1985), SSB1 (Jong e t al., 1987), NSR1 (Lee e t al., 1991) and GAR1 (Girard et al., 1992) possess the GAR domain which is rich in glycine and arginine residues. Biophysical analysis has shown that the GAR domain of the nucleolin destabilizes the structure of RNA duplexes in a nonsequence-dependent manner possibly to allow access of specific binding components (Ghisolfi et aL, 1992). The GAR-like motifs of the maize MA16 protein: R(G)nY(G)nR and RR(E/D)(G)nY(G)n, are more similar to the GAR domains of these nucleolar proteins than to those of the hnRNP proteins. Moreover, the results of the dendrogram showed a higher proximity of MA16 with the cluster of nucleolar proteins than with the nuclear hnRNP cluster. Whether or not these physical similarities are related to functional similarities, remains to be determined. In summary, the data obtained in this study should provide a starting point to address questions about the biological role of MA16. The wide distribution and accumulation pattem of MA16 in actively growing tissues together with its nucleolar localization in the DFC and GC suggest that the MA16 protein is probably involved in general growth phenomena in the plant and that it could play a specific role in pre-rRNA processing and/or ribosome assembly. We are currently in the process of testing these hypotheses. Experimental procedures Plant material Embryos of maize (Zea mays) pure inbred line W64A were dissected manually and used immediately after collection. Seedlings were dehydrated for 3 h ($1), 1 day ($2) and 3 days ($3) as previously described (Gomez et al., 1988). Nucleolar localization of the maize MA 16 protein 833 Microscopy Acknowledgements For immunolocalization, the immune serum obtained against the MA16 protein (Ludevid et a1.,1992) was used. An all-purpose fixative (80% ethanol, 3.5% formaldehyde, 5% acetic acid) was used for paraffin embedding. Sections from paraffin-embedded material were blocked with 3% goat serum in phosphate-buffer saline (PBS; 10 mM phospate, 150 mM NaCI, pH 7.4) for 30 min at 22°C, and incubated with anti-MA16 immune serum (diluted 1/1000), and pre-immune serum (diluted 11500); immunoreactivity was visualized by the Avidin-biotin complex (Vectastain Elite ABC Kit, Vector Burlingame, CA) using diaminobenzidine as substrate. Double labelling of the same cells was performed with calcofluor and by indirect immunostaining with the antiMA16 antibody. For in situ hybridization digoxigenin-labelled RNA probes were prepared according to the manufacturer's instructions (Boehringer Mannheim). In situ hybridization was performed as described by Jackson (1991). Sense and antisense probes were transcribed from the MA16 cDNA clone in the pBluescript SK+ vector (Stratagene). In all cases, no signal over background was observed using control sense-strand probes. For immunoelectronmicroscopy embryos of 30 and 50 d.a.p. and leaves of 6-day-old plantlets were cut in pieces of approximately 1 mm3 and fixed in 0.5% glutaraldehyde and 4% paraformaldehyde in phosphate buffer (20 mM, pH 7.3) for 2 h. After washing with phosphate buffer, the samples were dehydrated in ethanol series and embedded in LR-White resin polymerized at 60°C. Ultrathin sections (60-80 nm) were mounted on gold grids. The grids were floated on drops of blocking solution (2% eggalbumin in PBS containing 0.65 M NaCI) for 1 h, and then incubated with the rabbit antiserum against MA16 diluted 1:1000 O/N at 4°C. After washing with PBS, the sections were incubated with protein A coupled with colloidal gold (10 nm). The sections were stained with 2% aqueous uranyl acetate and Reynolds lead citrate and then examined on a Philips 301 electron microscope. Parallel controls with pre-immune serum were performed. We thank Drs M. Dolors Ludevid and Margarita Torrent for stimulating discussions and advice on subcellular fractionation and polysome isolation experiments. Drs M. Carmen RisueSo (CIB-CSIC, Madrid) and Carmen Lbpez-lglesias (Universitat de Barcelona) are thanked for helpful suggestions in immunoelectron microscopy, and the Departament de Microscbpia Electrbnica (Universitat de Barcelona), for the use of their facilities and technical assistance, We are grateful to Drs Pere Puigdemenech, Montserrat Bach and Stephen D. Jackson for reading, and constructive criticism of, the manuscdpt and Dr Claude Gigot (IBMP-CNRS, Strasbourg) for generously providing the maize H2NH2B antibodies used. M. Mar Albb was supported by a predoctoral fellowship from the Departament d'Ensenyament of the Generalitat de Catalunya. This work was supported by grants BIO91-0546 from Plan Nacional de Investigacibn Cientifica y Desarrollo Tecnolbgico and BIO2 92-0529 from the European Economic Community BIOTECH Program to M.P. Subcellular fractionation polysome isolation and protein gel blot analysis References Beebee, T.J.C. (1986) Nucleoli and predbosomal particles. In Nuclear Structures. Isolation and Characterization, Volume 7 (MacGillivray, A.J." and Birnie, G.D., eds). Butterworths, pp. 100-117. Bensen, R.J., Boyer, J.S. and Mullet, J.E. (1988) Water deficitinduced changes in abscisic acid, growth polysomes and translatable RNA in soybean hypocotyls. Plant Physiol. 88, 289-294. Bergeron, D., Beauseigle, D. and Bellemare, G. (1993) Sequence and expression of a gene encoding a protein with RNA-binding and glycine-rich domains in Brassica napus. Biochim. Biophys. Acta, 1216, 123-125. Borer, R.A., Lehner, C.F., Eppenberger, H.M. and Nigg, E.A. (1989) Major Nucleolar proteins shuttle between nucleus and cytoplasm. Cell, 56, 379-390. Bourbon, H. and Amalric, F. (1990) Nucleolin gene organization in rodents: highly conserved sequences within three of the 13 introns. Gene, 88, 187-198. Burd, C.G., Swanson, M.S., Gorliich, M. and Dreyfuss, G. Nuclei of 30 d.a.p, embryos were essentially obtained as described by Wurtzel et al. (1987). According to this method, the nuclei were recovered from the 81% Percoll interface. Cytoplasmic proteins from the supernatant of the Percoll cushion were precipitated with 15% TCA. Subnuclear fractions were obtained as described by Beebee (1986). Nuclei were resuspended in a solution containing 0.25 M sucrose and 10 mM HCI-Tds, pH 7.5, and sonicated (4x 20 sec bursts, Branson 250, with standard probe). After centdfugation on a 0.88 M sucrose cushion, three fractions were collected, the pellet, the interface and the supematant, that were further analysed by Western blot. Polyribesomes were isolated according to Torrent et a/. 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