CARYOLOGIA Vol. 56, no. 1

Vol. 56, no. 1: 31-35, 2003
CARYOLOGIA
NORs in Rhoeo (Commelinaceae) revisited
HIERONIM GOLCZYK* and ANDRZEJ JOACHIMIAK
Department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52, 31-044 Kraków, Poland.
Abstract - Nucleolar organizer regions were localized by silver staining in Rhoeo
spathacea (Swartz) Stearn. Our results strongly suggest that interstitially located
lightly stained chromosome segments observed in conventional preparations
and interpreted by previous authors as secondary constrictions are depleted of
active nucleolus organizing regions. There were no Ag-positive intercalary signals
within chromosome arms where lightly stained chromosome segments were
observed. Ag-NOR loci were found only at telomeric and centromeric chromosome domains, and colocalized with heterochromatin bands.
Key words: Ag-staining, chromosomes, heterochromatin, karyotype, NORs,
Rhoeo spathacea.
INTRODUCTION
Rhoeo spathacea (Swartz) Stearn. (= Rhoeo
discolor) has been a classical subject of cytological studies for about 70 years since the pioneering
works of BELLING (1927), DARLINGTON (1929)
and SAX (1931). It is widely believed that the
permanent structural heterozygosity of this
species (the lack of chromosome pairs and unusual mode of meiosis, with chromosome rings or
chains at the first division) is conditioned by a
series of reciprocal translocations between the
distal segments of all chromosomes (for review:
SATTERFIELD and MERTENS 1972). Most of the
data on karyotype structure in this species have
been obtained from observations of conventionally stained meiotic chromosomes (KATO 1930;
SAX 1931, 1935; COLEMAN 1941; LIN and PADDOCK 1973, 1978; LIN 1979). Besides numerous
non-interstitially located heterochromatic bands
(NATARAJAN and NATARAJAN 1972; STACK 1974;
PETTENATTI 1987; GOLCZYK and JOACHIMIAK
1999), no other coding and non-coding domains
have been localized within the karyotype of this
interesting species.
There are some reports on the localization of
nucleolar organizers (NORs) in Rhoeo, but only
* Corresponding author: fax ++48-12-4228107, e-mail:
[email protected]
from indirect observations in unrelated studies.
DARLINGTON (1929), BHADURI (1942) and LIN
and PADDOCK (1973) found several interstitially
located, weakly stained regions within some conventionally stained meiotic/mitotic chromosomes,
and interpreted them as secondary constrictions
(“NORs”). However, the constrictions localized
by LIN and PADDOCK (1973) significantly differ
from those detected by BHADURI (1942) and DARLINGTON (1929). On the other hand, S TACK
(1974), based on the reasonable generalization
that nucleolus organizers in plants are usually
associated with constitutive heterochromatin, has
questioned the insterstitial localization of NORs
in this species and suggested the presence of only
two terminal NOR loci on two submetacentric
chromosomes equipped with telomeric heterochromatin on the shorter arms.
Secondary constrictions may be viewed as a
state of arrest of condensation of the regions
bearing the nucleoli at prophase, that is, they can
be treated as chromosomal markers of active
NORs within conventionally stained preparations
(BUSH and SMETANA 1970). Although not all
interstitially located active NORs are marked by
a secondary constriction, all true secondary constrictions should be treated as active NOR sites.
Transcriptionally inactive rDNA clusters, if present, do not form secondary constrictions, and
satellites are not observed within the chromo-
32
GOLCZYK
and JOACHIMIAK
Fig. 2 – Karyotype of Rhoeo spathacea. Chromosomes
ordered according to their relative position within meiotic
rings/chains. Black – chromosome regions with the highest
frequency of LSSs (above 0.4).
Fig. 1 – Somatic Rhoeo metaphase chromosomes with clearly visible LSSs. Acetoorceine staining. Bar 10µm.
tional and C-banding methods (G OLCZYK and
JOACHIMIAK 1999). Floral buds were obtained from
the same donor plants. Root tips and anthers were
fixed in AA (glacial acetic acid and absolute alcohol,
1:3) for 2 h, stained in aceto-orcein (2% solution of
orcein in 45% acetic acid) at room temperature and
squashed in a drop of 45% acetic acid with no heating to minimize the risk of chromosome breaks. For
Ag-staining the material was fixed overnight refrigerated in FAA (50% alcohol, glacial acetic acid and
37% formaldehyde, 18:1:1), squashed and stained
according to Trude Schwarzacher and Karyobiology
Group (http://www.le.ac.uk/bl/phh4/methods/agnor.htm). The twentyfive best mitotic metaphases and
ten best meiotic chains were selected and captured in
a Lucia G system (Laboratory Imaging Ltd., Czech
Republic) or photographed. For C-banding the material was fixed in AA for 2 h and further treated
according to the schedule described by SCHWARZACHER et al. (1980).
somes bearing them (LENGEROVA and VYSKOT
2001). The intercalary localization of NORs in
Rhoeo suggested by the majority of previous
authors (l.c.) seems in disagreement with the lack
of intercalary heterochromatin (STACK 1974; PETTENATI 1987; GOLCZYK and JOACHIMIAK 1999),
the apparent lack of interstitial CMA-positive signals (JOACHIMIAK and GOLCZYK, unpbl. results)
and also the absence of stable types of satellited
chromosomes in conventionally stained mitotic
preparations (GOLCZYK and JOACHIMIAK 1999).
Thus it seems probable that the previously
described “secondary constrictions” in Rhoeo are
artifacts or else represent chromatin segments of
unknown structure but lacking active nucleolar
organizers. For the first time we use silver staining (Ag-staining), a simple technique specifically
detecting functional nucleolus organizers (CHEUNG et al. 1989) within the Rhoeo karyotype to
resolve this problem.
Data processing and presentation
Images were captured and processed using a CCD
camera and Lucia G. The chromosome lengths were
calculated as percentages of total karyotype length.
Karyotype analysis was performed on 10 complete
meiotic and 25 mitotic metaphases. Chromosome
types were identified and classified as previously
described (GOLCZYK and JOACHIMIAK 1999).
MATERIALS AND METHODS
Cytological methods
Roots were taken from cuttings of five different R.
spathacea plants we previously analyzed by conven-
Table 1 – Rhoeo spathacea, acetoorceine stained chromosome types (1-12) showing different number of LSSs within five chromosome regions (ChR) in mitosis (a) and meiosis (b).
1
ChR
a
1
6
2
11
3
1
4
11
5
1
Total 30
2
b
4
2
0
0
2
8
a
1
13
10
0
0
24
3
b
1
3
0
0
0
4
a
1
12
7
1
0
21
b
1
2
0
0
0
3
4
5
a b
3 1
17 1
1 0
11 2
1 0
33 4
a b
5 1
18 4
16 0
2 0
1 0
42 5
6
a
1
10
2
1
0
14
7
b
0
2
0
0
0
2
a
1
9
0
7
1
18
8
b
0
1
0
1
0
2
a
1
11
1
1
0
14
9
b
3
0
0
0
0
3
a
1
5
0
0
0
6
10
b
0
0
0
0
0
0
a
0
3
0
8
0
11
11
b
0
0
0
0
0
0
a
1
3
0
2
1
7
12
b
0
0
0
0
0
0
a b
0 0
11 2
2 0
1 0
0 0
14 2
NORs IN RHEO REVISITED
33
Fig. 3 – Chromosomes (A-D) and nuclei (E-H) of Rhoeo spathacea. A-C, silver-stained mitotic metaphase plates with two (A),
three (B), and five (C) distinct telomeric Ag-NOR signals. Note the large silver positive centromeric signal in B (arrowed) and other centric signals appearing as tiny double dots. D, C-banded mitotic metaphase plate. E-G, interphase nuclei from the root meristem showing different numbers of silver-stained nucleoli. H, mitotic prophase nucleus with large, collective nucleolus. Bars 10µm.
34
GOLCZYK
The lightly stained chromosome segments (LSSs)
we observed (see Introduction) were found in most
aceto-orcein stained metaphases. The LSS positions
within each analyzed chromosome were calculated as
the distance from the end of the longer chromosome
arm. Each chromosome type was divided into five
regions of equal length, the first region beginning at
the end of the longer arm (Fig. 2, Table 1). The expected vs. observed distribution of LSSs along the distinguished regions of chromosomes was tested with the
chi-square test for goodness of fit (SOKAL and ROHLF
1969). The expected distribution was an even one,
that is, an equal chance that an LSS would appear in
a given chromosome region.
RESULTS
We observed LSSs (Fig. 1) within all conventionally stained mitotic metaphases and in six
meiotic rings/chains. Within meiotic chromosomes these structures were not easy for distinction and they were less frequently observed
(Table 1); thus mitotic metaphases seem more
suitable for analysis of their distribution. Mitotic
LSSs were clearly unstable as to their number
and localization within chromosomes. Their chromosome positions are highly variable, so finally
we distinguished only some chromosome regions
characterized by relatively high (above 0.4) LSS
frequency (Fig. 2). The number of chromosomes
with LSSs in a given metaphase varied from 3 to
11, and the number of these structures within a
particular chromosome was from 1 to 4. All chromosome variants with “secondary constrictions”
described by previous authors (DARLINGTON
1929; BHADURI 1942; LIN and PADDOCK 1973)
were observed in our study, but some of them at
a very low frequency. The observed distribution
of LSSs differed significantly from the expected
even distribution (chi-square test = 176, s.s. = 4,
p < 0.0001). The pattern of departure from the
expected distribution points to the central
domains of the longer chromosome arms, where
an excess of LSSs is manifested.
All the Ag-positive signals were located at
chromosome termini and/or within centromeric
clusters of chromatin (Figs. 3A, B, C). There were
no positive interstitially located signals within
arms, where LSSs were frequently observed in
orcein-stained preparations. In mitotic chromosomes large signals were located mainly at telomeres, whereas centric signals were weak and
detected chiefly in the form of tiny double dots
and JOACHIMIAK
(Figs. 3A, B, C). The number of potentially active
nucleolus organizers in Rhoeo (identified within
different chromosome positions) seems relatively high; this may be additionally supported by
the observations of interphase nuclei (Figs. 3E, F,
G). In the majority of root-tip interphase cells
the number of silver-stained nucleoli was 1-6,
markedly exceeding STACK’s (1974) estimates but
in accordance with BHADURI (1942), who stated
that the maximum number of separate nucleoli
observed in Rhoeo root meristem is 6. In fact,
the strong tendency to nucleolar fusion in Rhoeo
(Figs. 3G, H) obscures the situation; nucleoli
could be even more abundant (BHADURI 1942).
Centromerically located Ag-NOR signals colocalized with previously described (GOLCZYK and
JOACHIMIAK 1999) centric blocks of heterochromatin, but in C-banded mitotic chromosomes
such heterochromatin clusters are markedly larger than the majority of centric Ag-positive signals
detected here (Fig. 3D). Thus the possibility that
heterochromatin in Rhoeo is agyrophilic per se
can be ruled out. Terminal Ag-NOR signals could
also be traced to the heterochromatin because
five terminally located segments of heterochromatin have been carefully localized within the
Rhoeo karyotype (GOLCZYK and JOACHIMIAK
1999). Unfortunately, exact identification of
NOR-bearing Rhoeo chromosomes by morphology is very difficult in our silver-stained preparations, and further studies are needed.
DISCUSSION
It is broadly accepted that Ag-NOR-positive
chromosome signals correspond to the locations
of active rRNA genes (CHEUNG et al. 1989), and
that the size and intensity of the Ag-stained areas
are related to their transcriptional activity
(MILLER et al. 1976; HOFGÄRTNER et al. 1979).
The observed exclusively proximal and distal
localization of Ag-stained regions strongly suggests that interstitially located LSSs are not true
secondary constrictions (structures organizing
the nucleolus). These lightly stained bands
observed by us and by previous authors (DARLINGTON 1929; BHADURI 1942; LIN and PADDOCK
1973) proved surprisingly unstable as to their
number and localization within different
metaphase plates. The nature of these bands
remains unknown; their distribution differs significantly from the expected random distribu-
NORs IN RHEO REVISITED
tion (see Results). Even if the LSSs are breaks
caused by the method used, their non-random
distribution and abundant occurrence within definite chromosome segments suggest increased
susceptibility of these segments to acidic treatment.
Acknowledgments – We are indebted to Dr. J.
Mitka for help and valuable discussion.
REFERENCES
BELLING J., 1927 – The attachement of chromosomes
at the reduction division in flowering plants. Journal of Genetics, 18: 177-205.
BHADURI P.N., 1942 – Cytological analysis of structural hybridity in Rhoeo discolor Hance. Journal
of Genetics, 44: 73-85.
BUSH H. and SMETANA K., 1970 – Nucleolar organizers, nucleolar size, shape and number. In: H.
Bush H and K. Smetana (Eds.), “The nucleolus”, pp. 115-159. Academic Press, New York
and London.
CHEUNG S.W., SUN L. and FEATHERSTONE T., 1989
– Visualization of NORs in relation to the precise
chromosomal localization of ribosomal RNA
genes. Cytogenetics and Cell Genetics, 50: 93-97.
COLEMAN L.C., 1941 – The relation of chromocenters
to the differential segments in Rhoeo discolor
Hance. American Journal of Botany, 28: 742-8.
DARLINGTON C.D., 1929 – Chromosome behaviour
and structural hybridity in the Tradescantiae.
Journal of Genetics, 21: 207-286.
GOLCZYK H. and JOACHIMIAK A., 1999 – Karyotype
structure and interphase chromatin organization
in Rhoeo spathacea (Sw.) Stearn (Commelinaceae). Acta Biologica Cracoviensia Series
Botanica, 41: 143-150.
HOFGÄRTNER F.J., KRONE W. and JAIN K., 1979 –
Correlated inhibition of ribosomal RNA synthesis
and silver staining by actinomycin D. Human
Genetics, 47: 329-333.
KATO K., 1930 – Cytological studies of pollen mother cells of Rhoeo discolor Hance, with special reference to the question of the mode of syndesis.
35
Memoirs of the Collegium of Science of Kyoto
Imperial University Series B, 5: 139-161.
LENGEROVA M. and VYSKOT B., 2001 – Sex chromatin and nucleolar analyses in Rumex acetosa L.
Protoplasma, 217: 147-153.
LIN Y.J., 1979 – Chromosome distribution and catenation in Rhoeo spathacea var. concolor. Chromosoma, 71: 109-127.
LIN Y.J. and PADDOCK E.F., 1973 – Ring-position
and frequency of adjacent distribution of meiotic
chromosomes in Rhoeo spathacea. American
Journal of Botany, 60: 685-690.
–, 1978 – Identification of complexes in a spontaneous triploid Rhoeo spathacea. Chromosoma,
67: 97-108.
MILLER O.J., MILLER D.A., DEV V.G., TANTRAVAHI
R. and CROCE C.M., 1976 – Expression of human
and suppression of mouse nucleolus organizer
activity in mouse-human somatic cell hybrids.
Proceedings of the National Academy of Sciences U.S.A., 73: 4531-4553.
NATARAJAN A.T. and NATARAJAN S., 1972 – The
heterochromatin of Rhoeo discolor. Hereditas,
72: 323-330.
PETTENATI M.J., 1987 – Giemsa C - banding of
Rhoeo (Commelinaceae). Genetica, 74: 219-224.
SATTERFIELD S.K. and MERTENS T.R., 1972 – Rhoeo
spathacea: A tool for teaching meiosis and mitosis.
Journal of Heredity, 63: 375-378.
SAX K., 1931 – Chromosome ring formation in
Rhoeo discolor. Cytologia, 3: 36-53.
–, 1935 – Chromosome structure in the meiotic chromosomes of Rhoeo discolor Hance. Journal of
Arnold Arboretum, 16: 216-224.
SCHWARZACHER T., AMBROS P. and SCHWEIZER D.,
1980 – Application of Giemsa banding to orchid
karyotype analysis. Plant Systematics and Evolution, 134: 293-297.
SOKAL R.R. and ROHLF F.J., 1969 – Biometry. The
principles and practice of statistics in biological
research. Freeman W.H. and Co., San Francisco.
STACK S.M., 1974 – Differential giemsa staining of
kinetochores and nucleolus organizer heterochromatin in mitotic chromosomes of higher plants.
Chromosoma, 47: 361-378.
Received June 15, 2002; accepted August 20, 2002