J. Cell Sd. 62, 171-176 (1983)
171
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FEULGEN BANDING OF HETEROCHROMATIN IN
PLANT CHROMOSOMES
G. E. MARKS
John Innes Institute, Colney Lane, Norwich NR4 7UH, England
SUMMARY
Feulgen bands can be obtained at the sites of constitutive heterochromatin in the chromosomes
of Anemone blanda, Fritillaria lanceolata and Scilla siberica, simply by means of a short or
extended acid hydrolysis. Extended hydrolysis gives positive bands in A. blanda and F. lanceolata
and negative bands in 5. siberica. Short hydrolysis gives no bands in A blanda and 5. siberica but
gives negative bands in F. lanceolata. The kind of Feulgen banding obtained is not correlated with
the type of base richness of the heterochromatin DNA; rather, it is probably due to differences in
the associated nucleoproteins.
INTRODUCTION
In the last decade or so, considerable attention has been given to the study of
constitutive heterochromatin, principally through the advent of chromosome banding
techniques utilizing Giemsa stain or fluorochromes. The preoccupation with Giemsa
has done little to elucidate the nature of heterochromatin because, with few exceptions
(Fiskesjo, 1974; LaCour, 1978; Sato, Kuroki & Ohta, 1979; Yen & Filion, 1977), it
stains all heterochromatin uniformly. Fluorochromes, however, differentiate between A + T and G + C-rich heterochromatin (reviewed by Schweizer, 1980).
Considering the interest shown in understanding the nature of heterochromatin and
the basis of chromosome banding, it is somewhat surprising, because of its known
specificity for DNA, that very little attention has been given to using the Feulgen
method in banding studies. This is especially so since it was demonstrated by
Yamasaki (1973) in Cyprepedium debile and by Ennis (1975) in two species of
Chilocorus that by extending acid hydrolysis it was possible to obtain both Feulgen
positive (F + ) and Feulgen negative (F~) bands corresponding, respectively, to G + C
and A + T-rich heterochromatin. Recently, D'Amato, Bianchi, Capineri & Marchi
(1981a) have demonstrated banding in unfixed plant metaphase chromosomes after
a conventional Feulgen staining procedure. F + bands were found in chromosomes of
Scilla siberica and F~ bands in chromosomes oiBrimeura amethistina, Ornithogalum
montanum and Vicia faba. Quinacrine staining gave reduced fluorescence in
5. siberica chromosomes (G + C-richness) and enhanced fluorescence in those of the
other species (A + T-richness). Subsequently, D'Amato, Capineri & Marchi (19816)
demonstrated F + bands in chromosomes of Buglossoidespurpurocaerulea, which also
show reduced quinacrine fluorescence (G + C-richness) both in fixed and unfixed
chromosomes. These results substantiate those obtained in Cyprepedium and
Chilocorus, which suggests a correlation between F + bands and G + C-richness and
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G. E. Marks
F~ bands and A + T-richness but, having established this possible correlation, it is
of particular importance to note that D'Amato et al. (1981a) also showed, from a
preliminary observation, that by controlling hydrolysis time, it was possible to change
F~ to F + bands in chromosomes of Ornithogalum montanum, a species known to have
A + T-rich heterochromatin (Vosa & Marchi, 1972).
The chromosomes of Anemone blanda are particularly well endowed with regions
of Giemsa staining heterochromatin (Marks & Schweizer, 1974). These regions give
enhanced quinacrine fluorescence (Schweizer, 1980) indicating that they are A + Trich heterochromatin. In a preliminary experiment with A. blanda involving extended
acid hydrolysis prior to Feulgen staining, it was found that F + bands were produced
in all the heterochromatic regions. As this result is contrary to results obtained with
A + T-rich heterochromatin in Cyprepedium and Chilocorus, it could mean that F~
bands at least are not indicative of A + T-richness. This possibility prompted a more
comprehensive investigation of the effects of both short and extended acid hydrolysis
on the Feulgen staining of heterochromatin in A blanda. Also included in the study
were Fritillaria lanceolata and S. siberica, two species that are known to have
predominantly A + T-rich and G + C-rich heterochromatin, respectively (Deumling
& Greilhuber, 1982; Schweizer, 1973, 1976, 1980; Timmis, Deumling & Ingle, 1975;
Vosa, 1973). The results obtained form the basis of this paper.
MATERIALS
AND
METHODS
Root-tips of A. blanda, F. lanceolata and 5. siberica were taken from plants grown in a cool
greenhouse, and pretreated in either 005 % or 0-025 % (w/v) colchicine solution for 4—6 h, according to species, then subsequently fixed overnight in 1:3 (v/v), acetic acid/ethanol. Roots for
Feulgen staining were hydrolysed in 5 M-HC1 at room temperature for 4min (short hydrolysis),
15 min (control, standard hydrolysis) and 60-100 min (extended hydrolysis). After hydrolysis, roots
were well washed in deionized water and stained in Feulgen reagent for l i h in the dark at room
temperature. After rinsing root-tips in SOz/water, squash preparations were made in 45 % acetic
acid and these were made permanent by removing the coverslip on solid COz, air drying and
mounting in Euparal. Giemsa preparations were made according to the schedule given by Marks
(1975).
RESULTS
AND
DISCUSSION
In the chromosomes of all three species studied, constitutive heterochromatin is
revealed as positive bands with Giemsa staining (Figs 1—3). With Feulgen staining
after conventional hydrolysis no banding is discernible in the chromosomes. However, banding of constitutive heterochromatin is obtained after both short and extended hydrolysis, appearing as either F + or F~ bands according to the treatment and/
or species concerned. In A blanda short hydrolysis gives no banding whereas extended hydrolysis gives F + bands, corresponding in position to those obtained with
Giemsa (Fig. 1). In 5. siberica, short hydrolysis gives F~ bands corresponding to
Giemsa bands (Fig. 2). InF. lanceolata short hydrolysis gives F~ bands and extended
hydrolysis F + bands that, with few exceptions, correspond to each other and to
Giemsa bands (exceptions are secondary constrictions, probably nucleolar organizing
regions) (Fig. 3).
Feulgen banding
173
i *i j i
1
2
3
Fig. 1. Chromosomes of A. blanda. Left-hand column, Giemsa-stained. Central column,
Feulgen-stained after short hydrolysis. Right-hand column, Feulgen-stained after extended hydrolysis. X1200.
Fig. 2. Chromosomes of S. siberica. Staining procedures and magnification as in Fig. 1.
Fig. 3. Chromosomes of F. lanceolata. Staining procedures and magnification as
in Fig. 1.
The intensity of Feulgen staining is directly proportional to the amount of apurinic
acid generated and retained in situ (Kjellstrand, 1980; Lessler, 1954), and this can be
affected by the following factors:
(1) The concentration of DNA per unit of chromosome.
(2) Base-pair composition.
(3) Accessibility of DNA to hydrolysis.
(4) The retention of apurinic acid in the chromosome matrix.
It is simplest to consider first those species, A. blanda and 5. siberica, that produce
bands only after extended hydrolysis, F + in the former and F~ in the latter. Banding
in these species cannot be solely the result of differences in DNA concentration,
because if this was so then banding would not depend on extended hydrolysis. Bostock
& Sumner (1978) have reviewed the evidence concerning the possibility that
chromosome bands are related to different amounts of DNA along the chromosome,
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G. E. Marks
from which it appears that, although such differences in DNA occur, these are
nevertheless insufficient to account for the staining reactions observed. Chromosome
banding, therefore, is not a consequence of differences in DNA concentration. The
results with Chilocorus (Ennis, 1975) indicate that there is an association between
base-pair composition and the type of Feulgen staining, i.e. G + C-rich
heterochromatin gives F + bands and A + T-richness gives F~ bands. In Cyprepedium
also, G + C-rich heterochromatin gives F + bands (Yamasaki, 1973). However, in the
species described here, the associations between types of Feulgen banding and basepair richness are reversed. Likewise, A + T-rich heterochromatin in V. faba
(Schweizer, 1980) also gives F + bands (Tempelaar, de Both & Versteegh, 1982).
Consequently, when the Chilocorus and plant species results are considered together,
there appears to be no correlation between the kind of base richness of heterochromatin DNA and the type of Feulgen banding. Indeed this conclusion is most evident
from the results with S. siberica andF. lanceolata. In the former species, comparison
of the present results (F~ bands) and those of D'Amato et al. (1981a) (F + bands)
shows that the kind of banding depends upon fixation; in the latter species the kind
of banding depends upon the extent of hydrolysis. Clearly, since these procedures
cannot alter the base richness of the heterochromatin, it must follow that base richness
per se does not determine the kind of Feulgen banding. This conclusion is in keeping
with the evidence of Rodman & Tahiliani (1973) who found no difference in the
depurination rates of adenine and guanine.
Since Feulgen banding does not appear to be conditioned by either DNA concentration or base composition, it follows that it may be due to differences between
heterochromatin and euchromatin in other DNA-associated components, which affect either the accessibility of DNA to hydrolysis or the retention of apurinic acid.
Rodman & Tahiliani (1973) used the Feulgen method to estimate relative amounts of
DNA retained in the heterochromatin of mouse chromosomes after preparations had
been pretreated with buffers and hot saline. They also found F + and F~ bands
corresponding to A + T and G + C-rich heterochromatin. Since the effects of the
denaturing treatments used on mouse chromosomes are similar to those of extended
acid hydrolysis in that they both remove DNA from chromatin, it follows that the
results with mouse chromosomes are comparable to those described here. Rodman &
Tahiliani (1973) consider banding to reflect the regional distribution of two kinds of
nucleoproteins, those retaining aggregates of DNA to give F + bands and those that
are more readily removed by hydrolysis, leaving the DNA unprotected and thus
giving F~ bands. A similar assumption concerning associated nucleoproteins would
explain the results obtained with A. blanda and 5. siberica. In the former, the
heterochromatin relative to euchromatin is better protected from the loss of apurinic
acid after extended hydrolysis, thus giving F + bands, whereas in the latter species the
contrary situation would apply, to give F~ bands. On this assumption, the rate of
depurination in the unfixed heterochromatin of 5. siberica must be greater than in the
euchromatin, to give F + bands after normal hydrolysis as found by D'Amato et al.
(1981a). Fixation, therefore, seems to alter the protective nature of the
nucleoproteins.
Feulgen banding
175
+
If we now consider banding in F. lanceolata in which bands can be either F or F~
depending upon the extent of hydrolysis, it is also possible to explain the results by
assuming that the heterochromatin DNA is protected by its associated nucleoprotein.
In this case, the protection produces a lag in the onset or reduces the rate of hydrolysis,
so that there would be relatively less depurination in the heterochromatin at first,
giving F~ bands, while with extended hydrolysis depurination would continue longer
in heterochromatin to give F + bands.
F~ banding of heterochromatin in Fritillaria was amongst the first demonstrations
of chromosome banding (Darlington & La Cour, 1941). In the method employed,
banding was dependent upon growing the meristems at 0—3 °C for some days prior to
fixation, followed by the normal Feulgen procedure. The present results show that
F~ banding can be produced without cold treatment, simply by reducing the
hydrolysis time. Given that the differential understaining of heterochromatin with
Feulgen is due to a protective nucleoprotein, then it follows that the effect of cold
treatment is to enhance this protection through altering the physicochemical relationship between the DNA and protein, such that the effect persists under the conditions
of normal hydrolysis.
That there are, in fact, many 'species' of heterochromatin is clear from information
obtained from studies relating to its composition, behaviour and role in the genotype
(Bostock & Sumner, 1978). The results presented here suggest that this diversity of
heterochromatin — and also the differences between euchromatin and heterochromatin in their staining reactions — is not a simple direct consequence of differences
between species of DNA, but rather it is due to differences in other components of
chromatin, probably the nucleoproteins.
The author is indebted to Dr Dieter Schweizer for reading the manuscript and for his valuable
comments.
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{Received 25 November 1982-Accepted 7 January 1983)