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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
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