J. Cell Sci. 6, 87-107 (1970)
Printed in Great Britain
87
TEMPERATURE-DEPENDENT FUNCTIONAL
STRUCTURES IN THE POLYTENE
CHROMOSOMES OF PHASEOLUS, WITH
SPECIAL REFERENCE TO THE NUCLEOLUS
ORGANIZERS
W. NAGL*
Max-Planck-Institut
filr Pflanzengenetik, 6802 Ladenburg-Rosenhof, West Germany
SUMMARY
During the endopolyploidization of the suspensor cells of Phaseolus coccineus and P. vulgaris,
polytene chromosomes and an important nucleolar apparatus develop within the nuclei. The
nucleolus-associated regions of the four nucleolus-organizing chromosomes and the regions of
the secondary constrictions (intranucleolar chromatin threads) show a temperature-dependent,
reversible variability in their structure. They split up and are dispersed at optimal temperatures,
and they are compact and partly banded at too low or too high ones. Many nucleolar extrusions,
and additional nucleoli which originate on distended elements of the heterochromatic parts of
different giant chromosomes, occur at temperatures higher than 15 CC.
The polytene chromosomes of P. coccineus are able to form lateral loops projecting at right
angles to their axes, shortly after temperature elevation. This occurs by the uncoiling of the
superficial chromomeres. Several loops synthesize droplets and, moreover, they form
nucleolus-like bodies in a hitherto unknown manner. The duration of the lampbrush state is
about 3-4 h.
Cytochemical staining methods indicate the presence of DNA in all parts of the polytene
chromosomes, including the intranucleolar secondary constriction and the lateral loops, and the
presence of RNA in all types of nucleoli and nucleolus-like bodies.
The structural modifications described are compared with the structure of salivary gland
chromosomes of Diptera and lampbrush chromosomes in oocytes and spermatocytes. It is
suggested that the structural changes are an expression of temperature-dependent changes in
gene activity.
INTRODUCTION
Polytene chromosomes occur not only in animals, but also in Angiospermae
(Tschermak-Woess, 1956, 1957; Hasitschka-Jenschke, 1959, 1962; Enzenberg, 1961;
Nagl, 1962 a, b\ Erbrich, 1965). In some species of higher plants polytene chromosomes,
which occur within the ovule, are found only occasionally (review by TschermakWoess, 1963), but in Rhinanthus and Phaseolus they are always present (TschermakWoess, 1957; Nagl, 1962a, b, 1969a). The giant chromosomes of Rhinanthus are,
however, individually indistinguishable because of their granular, unbanded appearance, and the most favourable material for the study of plant polytene chromosomes
therefore appears to be the suspensor of the legume Phaseolus. In this genus the highly
• Present address: Institute of Botany, The University of Vienna, 1030 Wien, Austria.
88
W. Nagl
endopolyploid basal cells of the suspensor contain 22 unpaired giant chromosomes,
which are individually distinguishable, at least in part, on the basis of their different
lengths, the different length ratios of their arms, and the specific distribution of euand hetero-chromatic regions (Nagl, 19626, 1965, 1967). Moreover, different structural
modifications in the form of local swellings and lateral projections were found in the
species P. coccineus (Nagl, 1967), but the relationship of these to internal and external
factors remained unclear.
Temperature is an important factor in plant growth, and it influences the gross
morphology of the polytene chromosomes in Phaseolus vulgaris (Nagl, 1969a). In
Diptera, in addition to a similar effect of this kind (Bridges, 1936; Schultz, 1936;
Wolf, 1957; Bier, 1958, 1959), a change of the puffing pattern was observed following
temperature shocks (Berendes, van Breugel & Holt, 1965; Berendes, 1968). A study
was therefore made of the effect of growth temperature and of temperature shocks on
the morphology of the polytene chromosomes of Phaseolus. The results are presented
in this paper. Particular regard was given to the nucleolus-organizing chromosomes
and to the occurrence of additional nucleoli and nucleolus-like bodies, because these
give a morphological indication of the degree of nuclear activity.
MATERIALS AND METHODS
All studies and experiments were performed with phytotron-cultivated plants. The standard
conditions were the following: a light-dark rhythm of 13:11 h, 6 5 % relative humidity, and a
temperature of 27 °C in the light and 22 °C in the dark. In the experiments only the temperature
was changed. One phytochamber was used throughout to maintain the standard conditions for
controls. For each experiment 20 plants of Phaseolus vulgaris (variety 'Hild's Marona') and
5 plants of Phaseolus coccineus (var. coccineus; seeds from the Botanical Garden of the University
of Vienna) were used. Most of the studies were made with ovules of pods of a length between
8 and 10 cm in P . vulgaris, and of 15 and 18 cm in P. coccineus. At this stage the cotyledons of
the embryo occupy half of the embryo-sac. The nuclei examined for structural modifications of
the polytene chromosomes showed a degree of polyploidy of 1024*, 2048*, or 4096.Y, estimated
from volume determinations (compare Nagl, 1962a, b) and by microphotometric measurements
of Feulgen-DNA (W. Nagl, unpublished).
The ovules were halved, and the suspensor—lying between the micropyle and the embryo—
was taken out and fixed in ethanol/acetic acid (3/1) at 4 °C for 24 h. Other fixatives are unfavourable, as they do not allow squash preparations. The chromosomes are too large for paraffin
sections to be useful. However, fixation in 10 % neutral formalin was carried out if proteins were
to be stained. The ethanol/acetic acid-fixed cells were treated with 45 % acetic acid at 22 °C for
3—6 h, squashed and examined under a phase-contrast microscope. The quality of this preparative technique was tested by comparison with living cells of lower endopolyploidy.
Cytochemical methods were used for the localization of DNA, proteins and RNA. DNA was
detected using the Feulgen reaction; the hydrolysis was carried out with N HC1 (60 C C, 10 min)
after pectinase treatment (compare Nagl, 1967), and sometimes with 1 0 % perchloric acid
(20 °C, 15 h); pectinase of Serva (Heidelberg), and />-rosaniline, free of acridine (Chroma-Ges.,
Stuttgart) were used. Basic proteins (histones) of the chromosomes were stained according to
the method of Alfert & Geschwind (1953); for this, the formalin-fixed cells were hydrolysed
with trichloroacetic acid (5 %, 100 °C, 15 min), attached to the slides with Celloidin (1 %), and
stained with fast green F C F (National Aniline Division, New York) at pH 8-2. The methyl
green-pyronin method of Brachet (1953) was used for the detection of RNA in nucleoli and
nucleolus-like bodies (stains from G. Gurr Ltd, London); controls were treated with ribonuclease (Serva, Heidelberg) for 3 h at pH 7 0 (3 mg/ml, 38 °C). Some cells were stained with fast
gTeen at pH 5 0 , and acid fuchsin at pH 7-0 for other proteins, including acid proteins. For
Polytene chromosomes in Phaseolus
89
studies of the gross morphology of the polytene chromosomes, the cells were hydrolysed with
N HC1 at 55 CC for 15 min and stained with pyronin (Nagl, 1967), or hydrolysed for 30 min and
stained with 005 % toluidine blue O (Merck A. G., Darmstadt) at pH 4-0 for 1 h.
Unless otherwise stated, drawings and photographs were made from unstained preparations
using phase-contrast.
RESULTS
Development and gross morphology of the suspensor nuclei
During the development of the suspensor three stages are distinguishable: (a) the
proliferation stage, which ends with a pro-embryo composed of diploid cells; (b) the
differentiation stage, during which the suspensor grows endomitotically, and the
embryo mitotically; and (c) the functional stage, during which the mature suspensor
probably nourishes the rapidly growing embryo, especially the cotyledons. When the
cotyledons reach the chalazal end of the embryo-sac, the suspensor cells are crushed
after a short degeneration period.
The development of the polytene chromosomes was studied in the basal cells of the
suspensor during stage (b). The duration of this was found to be about one week, under
the standard conditions.
The diploid and low endopolyploid nuclei possess some small chromocentres (and
endochromocentres) within a fine euchromatic background. Some of the heterochromatic areas are associated with the spherical nucleolus, evidently being the nucleolus
organizers and the satellites (the diploid complement contains 4 nucleolus-organizing
chromosomes). During the further endopolyploidization the endochromocentres enlarge, and the euchromatic elements join to form granular parts of the future giant
chromosomes (see Nagl, 19626), provided no mitosis interrupts the endopolyploidization process (Nagl, 1969c). Finally, the whole chromatin of the nucleus is arranged
in 22 polytene chromosomes (Fig. 4). They have characteristic lengths, showing a
characteristic pattern of their eu- and hetero-chromatic portions (compare the idiogram of Nagl, 1967). At optimal temperatures, the euchromatic regions of the chromosomes have a granular appearance, but cooling of the plants results in the appearance of a
banding pattern (Nagl, 1969a). The four polytene nucleolus-organizing chromosomes
are the most easily identified ones (Nagl, 1965).
The nucleolus also enlarges significantly during nuclear growth. It persists during
endomitosis, which is of the common angiosperm-type (Nagl, 1965, 1967). As soon as
the polytene chromosomes become distinct, the hitherto spherical nucleolus elongates,
and it becomes very irregularly shaped at higher degrees of endopolyploidy, with many
lobes (Fig. 5). Within the nucleoli of all stages bright vacuoles can be seen. Chromatic
material, evidently forming the secondary constriction of the nucleolus-organizing
chromosomes, is perceptible in some of the vacuoles. The total connexion between the
nucleolus organizers and the satellites is commonly visible only after hydrolysis and
staining. It must be noted that in some cells the nucleolus divides into 2 or more parts.
In this case, every nucleolar fragment is in connexion with one, or one pair, of chromosomes, the larger pieces always with those of type I (see Nagl, 1965, 1967).
o,o
W. Nagl
The effect of growth temperature on the nucleolus-associated regions of the polyteve
chromosomes
The structure of the nucleolus-associated regions of the polytene chromosomes, i.e.
the nucleolus organizers and the satellites, was studied at three temperature levels:
27722 °C, i5°/i2 CC, and i2°/8 °C (the first value denotes the light period, the second
the dark period).
At 27722 °C, the nucleolus-associated regions split up into many threads of diminishing thickness, running partly on the surface of the nucleolus, and penetrating
partly along vacuoles (Fig. 6). The satellites are shaped like a spider (Fig. 7), or they
may be fragmented into ramified or spherical patches, lying on the surface of the
nucleolus (Fig. 5). If the temperature is lowered to 15 °C or 12 °C for 2 days or longer,
the opened state of the nucleolus-associated regions is reduced, and the degree of
ramification is less than at higher temperatures. A significant change in the structure
occurs at i2°/8 °C. Under these conditions most of the nuclei have condensed nucleolus
organizers, and the satellites become compact and spherical (Figs. 8, 9). This structural
change is reversible on returning the plants to one of the higher temperature levels for
some hours. It seems that the nucleolus organizers and satellites respond more quickly
to temperature alterations than the other parts of the chromosome complement, and
that the reaction to raising the temperature occurs more rapidly than does that to
lowering it. Possibly only the intranucleolar chromatin is more sensitive to those
influences.
The effect of temperature on the main nucleolus and the intranucleolar chromatin
The size and structure of the nucleolus, and the appearance of the intranucleolar
chromatin (i.e. the secondary constrictions of the four nucleolus-organizing polytene
chromosomes) were studied in plants kept for 2 days or longer at the 3 temperature
levels noted above. In addition, temperature shocks of 35 °C were given for 4 h, and of
45°-5O °C for 2 h.
The size of the main nucleoli increases with temperature, except at extreme levels.
The nucleoli in plants cooled at i2°/8 °C for 2 days are unexpectedly large, occasionally
showing extrusions along the chromosomes. It seems that material is squeezed out by
the condensing chromosomes and unites with the nucleolus. If the chilling time is
extended beyond 10 days the nucleolus shrinks to a spherical structure of less than half
the normal size. The nucleolar structure, characterized by a dense granular matrix and
bright vacuoles at optimal temperatures, changes, with apparent loss of material, and
at the end the nucleoli look like empty vacuoles. By a careful warming of the plants the
structure reverts to the original form. First, after some minutes, dark spots become
visible within the nucleolus. Those spots and the nucleolus itself increase in size and,
after some hours, the original picture is restored. On the other hand, there is a constant
increase of the nucleolar size between 12 °C and 27 °C. Under the standard conditions
(27722 °C), the nucleoli produce many lobes and extrusions, and many spherical
particles are extruded as free nucleoli into the karyoplasm. A temperature shock of
35 °C for 4 h leads to the extrusion of numerous particles (Fig. 17). These extrusions
Polytene chromosomes in Phaseolus
91
again extrude material, lying as free bodies in the nucleus. Several vacuoles of the main
nucleolus join to form large central vacuoles. Finally, a temperature shock of 45 °C or
50 °C for 2 h causes the disappearance of the granular cortex of the main nueleoli,
Nucleolus
Vacuole
Centromere
Satellite —
Secondary
constriction
Extrusion X°
3S°C
10//m
45 °C
Fig. 1. P. cocdneus and P. vulgaris, suspensor. Semi-diagrammatic figures of the nucleolus organizer, the secondary constriction, and the satellite of polytene chromosome I at
different temperatures. At 12 °C (day) and 8 °C (night), and following a temperature
shock of 45 °C, the chromosomal elements involved in the formation of the nucleolus
are condensed; at 27°/22 °C, and following a shock of 35 °C, they are dispersed
('puffed'). Note the different appearance of the secondary constriction at all temperatures. The right arm of the chromosome and the nucleolus are incompletely
drawn.
which become very translucent and small. The nueleoli again appear as empty
vacuoles. The effect of recooling was not investigated, but the nueleoli of plants which
were allowed to grow at 2']°J22 °C for some days following a temperature shock showed
the same structure as those without a shock treatment.
The intranucleolar chromatin behaves in a similar manner to the nucleolus-
92
W. Nagl
associated regions of the polytene chromosomes. This chromatin, which constitutes the
fibrils of the polytene secondary constrictions, is recognizable as a fibrillar connexion
between the nucleolus organizers and the satellites following hydrolysis with N HC1,
and it is visible in the form of threads within intranucleolar vacuoles in unhydrolysed
preparations. If the temperature is low (i2°/8 °C), these threads are thick and compact
(Fig. 12). At higher temperatures (i5°/i2 °C and 27°/22 °C) the threads separate into
several thinner fibrils showing a fuzzy appearance (Fig. 13). Four hours after raising
the temperature to 35 °C the intranucleolar chromatin has a disperse-chromomeric
appearance (Fig. 14). This may be the obvious expression of a puffed state of this
chromatin. On the other hand, a temperature shock of 45 °C or 50 °C for 2 h induces
an entirely different structure. A marked condensation of the disperse chromatin to a
single thread per nucleolus-organizing chromosome occurs. These threads are mostly
banded (Fig. 15). High temperatures, therefore, change the structure of the nucleolusrelated parts of the polytene chromosomes in a similar way as does low temperature.
The structural changes described are reversible within a period of some hours; they
are summarized in Fig. 1.
In all experiments some plants were found which did not respond in the same way,
exhibiting no significant structural change of the nucleolus-organizing chromosomes.
The explanation of this requires further information about the relationship between
chromosomal structure and function, which is not at present available.
A temperature-induced lampbrush state of the polytene chromosomes
In P. coccineus a conspicuous structural change of the polytene chromosomes occurred after a temperature elevation from i2°/8 °C to 22 °C, or from 22°/i5°C to
300 C (and also at other temperature levels) in over half of some hundreds of cells
examined. More rarely, a similar structural change was observed after the increase of
the temperature correlated with the onset of day within the phytotrons. The suspensor
cells were fixed at intervals of 10 min during the first hour after temperature elevation,
and at every full hour subsequently up to 5 h.
During the first 20 min after temperature increase, all the polytene chromosomes of
the temperature-sensitive cells develop narrow loops, at right angles to their axes, first
in distinct euchromatic regions (Fig. 2), later along the whole chromosome length, so
that the loops finally appear as a dense veil of processes radiating from the chromosomes (Figs. 19, 21) and forming a dense fibrillar network within the nuclei (Fig. 18).
The analysis of the loops is possible only after isolating the individual polytene chromosomes by squashing them out of the nuclei. In such preparations it can be seen that
the chromosomes split up distally into fibrils with a simple structure; these fibrils are
evidently oligotene fibres and single chromosomes of the polytene bundle. The lateral
projections show a duplex nature and are closed at their ends, i.e. they are real loops
(Figs. 2, 22). The length of the loops varies from 2 to 25 /tm (their total length is double
this), and their diameter varies from approximately 0-25 to 0-40 /tm. The duration of
this temperature-induced lampbrush state amounts to 2 or 3 h; projections from
heterochromatic portions, not identified as loops, are recognizable for longer. Owing
to the difficulty in distinguishing the individual giant chromosomes during the lamp-
Polytene chromosomes in Phaseolus
93
brush state, it has not so far been possible to distinguish between structures which are a
consequence of the rapid development of the loops, and those which may be an expression of different activity in specific chromosomal loci. For the present it seems
certain that the loops start by uncoiling of the outer chromomeres of the polytene
chromosomes, because the thickness of the chromosome axes corresponds to the number of loops occurring. About 4 h after the onset of the temperature increase, most of
the loops are retracted (Fig. 20).
10/<m
Fig. 2. Camera lucida drawing of the right arm of polytene c h r o m o s o m e I I I of P . coccineus in the early l a m p b r u s h state (10 m i n after t e m p e r a t u r e increase from 15° to
30 °C); optical longitudinal-section.
Fig. 3. Schematic diagram to illustrate the development of a nucleolus-like b o d y on a
network formed b y the loops of a suspensor polytene chromosome of P. coccineus
(longitudinal section t h r o u g h the polytene c h r o m o s o m e ; pc — diameter of the polytene chromosome, of which only the outer four sister chromosomes are d r a w n ) : A, a
loose b a n d consisting of sister c h r o m o m e r e s ; B , the outermost c h r o m o m e r e uncoils to
form a loop; c, the end of the loop forms a network and starts the synthesis of droplets,
while a second c h r o m o m e r e of the same band is uncoiling; D, the synthesized droplets
are joining to form a nucleolus-like b o d y ; E, a nucleolus-like b o d y is finished; F , the
nucleolus-like body (vacuolated) disengages completely from the n e t w o r k ; and G, t h e
almost restored b a n d .
94
W. Nagl
A morphologically conspicuous phenomenon indicating a high chromosomal
activity is the production of hundreds of droplets and particles by these lampbrush
polytene chromosomes, 20-40 min after their formation (Fig. 24). Moreover, the final
parts of some loops undergo a transformation to spherical networks, which become
increasingly loaded with droplets (Fig. 25). Evidently, the droplets join to form dense,
nucleolus-like bodies, the magnitude of which is generally smaller than that of the
' additional nucleoli' dealt with in the next section. They never produce extrusions, but
disengage themselves completely from the synthesizing loops. There remain behind
only the empty networks (Fig. 26). This behaviour of loops is shown schematically in
Fig. 3. The droplets and nucleolus-like bodies migrate to the nuclear boundary and
disappear in a short time.
The formation of loops is not inducible in every cell or plant, but in only about 60%.
Evidently more factors are involved in the structural change of polytene chromosomes
than temperature alone.
Nucleolar extrusions and additional nucleoli
Nucleolar extrusions are spherical particles, ejected from the main and additional
nucleoli (Figs. 16, 17). The term 'additional nucleolus' is used in this paper for
nucleolar bodies seeming to originate on heterochromatic elements of the polytene
chromosomes at sites other than the nucleolus organizers (Figs. 10, 11). Both the extrusions and additional nucleoli are vacuolated like the main nucleolus. Chromatic dots
or filaments are visible within some of the vacuoles of additional nucleoli, indicating
that the chromatin is not in the condensed state (Fig. 11). The number of nucleolar
extrusions and also that of additional nucleoli increases from 1 to 3 in nuclei of cooled
plants up to 20-40 at optimal temperatures.
The loci where additional nucleoli were found are as follows. At temperatures between 150 and 35°C, one pair of polytene chromosomes in P. vulgaris always possess
additional nucleoli on their distal heterochromatic band. This pair of chromosomes is
the one which was found to be in the puffed state in most of the cells (Nagl, 19696).
The possibility that these are a third pair of nucleolus-organizing chromosomes is
improbable, because their additional nucleoli unite with the main nucleolus very infrequently (in less than 1 % of cells). Other polytene chromosomes of both species,
among them also the nucleolus-organizing chromosomes, possess additional nucleoli
on the proximal heterochromatin (i.e. the region of the centromere), or on any band.
The additional nucleolus was seen suspended on a heterochromatic filament originating from a heterochromatic portion of the chromosome in several other cells (Fig. 10).
Staining properties of the polytene chromosomes and nucleoli
The Feulgen reaction indicates that all parts of the polytene chromosomes contain
DNA, as does the secondary constriction. This, the intranucleolar chromatin, is more
intensely stained the more it is condensed. The same is valid for staining of basic
proteins with fast green at pH 8-2. It may be assumed, therefore, that the secondary
constriction consists of a DNA-histone-complex, as do the other parts of the chromosomes. The loops of the lampbrush state are not individually visible in Feulgen- or'
Polytene chromosomes in Phaseolus
95
fast green-stained preparations. In the former, the chromosomes possess a pink
fibrillar halo, indicating that the loops contain DNA. The individual loops are not
recognizable because the matrix is dissolved by hydrolysis. On the other hand, the
formalin-fast green method preserves too much of the cytoplasm, thereby preventing
detailed analysis of the polytene chromosomes. An appearance comparable to that of
Feulgen-stained lampbrush polytene chromosomes, but somewhat more distinct, is
given by staining the hydrolysed cells with toluidine blue (Fig. 21).
The RNA content of nucleoli was tested with methyl green-pyronin in combination
with ribonuclease. This method does not preserve the chromosomal structure very
well, since swelling occurs. The polytene chromosomes stain violet, or, in several
preparations, in a blue and violet pattern of banding. Following the treatment with
ribonuclease they stain green-blue. The main nucleoli and additional nucleoli of cells
not treated with ribonuclease stain red, but the droplets and nucleolus-like bodies of
the lampbrush stage stain only pink. If the cells are pretreated with ribonuclease, all
the nucleoli and nucleolus-like particles are preserved in form, but they appear to be
unstained. It was generally noticed that the nucleolar extrusions and nucleolus-like
bodies stain intensely with fast green at pH 5-0, and with acid fuchsin at pH 7-0. These
reactions may indicate high contents of proteins, especially the acid ones. The main
nucleolus, on the contrary, stains more intensely with fast green at pH 8-2, which may
signify a high content of basic proteins.
Structural changes during the degeneration of the suspensor cells
All the structures described differ significantly from those seen at the onset of degeneration of the suspensor cells. When this occurs, the nucleoli dissolve, and the
polytene chromosomes disintegrate into longitudinal fibrils, which are evidently the
single sister chromosomes which formed the polytene bundles (Fig. 23). Sometimes
they degenerate to pycnotic elements. Of course, the structural changes occurring in
degenerating cells are in no way reversible.
DISCUSSION
The influence of temperature on the gross morphology of polytene chromosomes has
been known since the early studies of Diptera (Bridges, 1936; Schultz, 1936; Wolf,
1957; Bier, 1958, 1959)- The chromosomes are more compact at low temperatures,
and they are more diffuse at higher ones. Recently, a similar effect has been found in the
polytene chromosomes of the suspensor of P. vulgaris (Nagl, 1969 a). A more specific
effect was obtained with the aid of temperature shocks in Drosophila and Rhynchosciara:
the induction of puffs, and the increased RNA synthesis in these puffs (Berendes
et al. 1965; Pavan, 1965; Berendes, 1968). This gives evidence of temperaturedependent gene activity.
In the course of earlier investigations in Phaseolus an inexplicable structural
variability of the giant chromosomes was noted (Nagl, 1965, 1967). The aim of the
present study was to demonstrate that one of the factors influencing the detailed
structure of these plant polytene chromosomes may also be temperature. The nucleolus-
96
W. Nagl
organizing chromosomes have been found to be the most sensitive. At optimal temperatures, especially following a temperature shock of 35 °C, a 'puffing' of the
intranucleolar and nucleolus-associated chromatin occurs, whereas this is extremely
condensed at too low or too high temperatures. Besides these reversible structural
modifications, the size of the nucleolus changes with temperature: in general, it is
largest at optimal temperature (compare Vogt-Kohne (1961) for Diptera), and in these
conditions the extrusion of numerous particles is recognizable. This extrusion phenomenon has also been observed in other nuclei and interpreted as evidence of nuclear
activity (Gottschalk, 1951; Tschermak-Woess & Enzenberg-Kunz, 1965; Johnson &
Jones, 1967; Kohlenbach, 1967). Since the injection of actinomycin D into the embryosac leads to a condensation of nucleolus-organizing polytene chromosomes and to a
diminution of the nucleolar size similar to that produced by adverse temperatures
(W. Nagl, unpublished observations), and since it is known that the synthetic activity of
condensed chromatin is much less than that of diffuse chromatin, we conclude that
there are temperature-dependent functional structures in the polytene chromosomes.
The formation of the nucleolus seems to be a result of the activity of the nucleolus
organizers (McClintock, 1934) and of the intranucleolar secondary constrictions
(Heitz, 1931; Kaufmann, 1934; Kahn, 1962; Hsu, Brinkely & Arrighi, 1967). The
occurrence of conspicuous chromatin structures within nucleoli has been reported also
in other cells (Granboulan & Granboulan, 1964; Lettre, Siebs & Paweletz, 1966; La
Cour, 1966), especially from salivary gland cells of Diptera (Heitz & Bauer, 1933;
Kaufmann, 1938; Mechelke, 1953; Beermann, i960; Rodman, 1968); a single exception was noted in Smittia, in which the chromatin of the nucleolus organizer is not
distinct from that in other bands (Jacob & Sirlin, 1964).
A specific' puffing' was hitherto found in only one pair of the polytene chromosomes
of Phaseolus (Nagl, 19696). However, there are additional nucleoli at many chromosomal sites other than the nucleolus organizers. These particles may indicate a synthesizing or storing function of several loci. Whether or not they are comparable with
the nucleolar bodies visible in other cells, and, particularly, in dipteran giant chromosomes (see Beermann, i960; Swift, 1962; Jacob & Sirlin, 1963; Gabrusewycz-Garcia &
Kleinfeld, 1966; Pelling & Beermann, 1966), cannot be decided at present. Like the
latter, they originate on heterochromatic parts of the polytene chromosomes, but evidently on loosened fibrils, because they possess threads or granules of intranucleolar
chromatin (compare Pavan, 1965; Gabrusewycz-Garcia & Kleinfeld, 1966).
A very conspicuous functional structure is inducible within the greater part of the
suspensor cell nuclei of P. coccineus by a temperature increase of about 15 °C. After
this, the whole polytene complement assumes a lampbrush state for a period of 3-4 h.
Generally, lampbrush chromosomes are meiotic chromosomes with projecting lateral
loops, and probably occur in all oocytes (Gall, 1954; Callan, 1957, 1966; Kunz, 1967)
and spermatocytes (Ris, 1945; Nebel & Hackett, 1961; Meyer, Hess & Beermann,
1961). Like the puffs of polytene chromosomes, the loops are interpreted as an uncoiling of the chromosome fibres, which are in a condensed state within the chromomeres (Callan, 1963; Gall, 1956). The hitherto described meiotic lampbrush
chromosomes of plants have a fuzzy appearance, but loops were not identified (Grun,
Polytene chromosomes in Phaseolus
97
1958; Lu, 1967; Peveling, 1967). The polytene chromosomes of P. coccineus, on the
contrary, develop distinct loops, evidently by the uncoiling of the superficial chromomeres. It seems that there is no essential difference between the chemical composition
of the loops in Phaseolus and in animals. The Feulgen reaction indicates DNA in the
loops of the suspensor chromosomes and the presence of DNA has also been demonstrated for the loops in oocytes (Alfert, 1954; Callan & Macgregor, 1958). The loops in
Phaseolus produce nucleolus-like bodies and droplets, believed to contain RNA on the
basis of the methyl green-pyronin method; RNA was also demonstrated in the matrix
of animal loops (Gall, 1954; Gall & Callan, 1962; Macgregor & Callan, 1962). However, more studies will be necessary to elucidate the function of this lampbrush state of
the plant polytene chromosomes. For the present, it seems certain that the loops are
not an artefact, and they are distinct, for example, from the fuzzy appearance of dipteran giant chromosomes following NaOH treatment (Painter, 1941). Apart from the
fact that they have been seen after good fixation, the polytene chromosomes of Phaseolus
disintegrate into longitudinal fibrils during degeneration (Fig. 23) and following chemical treatments (W. Nagl, unpublished).
The fact that the structures described are not inducible in all nuclei might be explained by genetical differences between different plants, and by physiological and
developmental differences between single cells. Moreover, temperature may be one factor
only within a very complex regulation system. A high degree of variability of nuclear
structure without obvious cause is one of the unexplained phenomena in the study of
nuclei in general (Tschermak-Woess, 1963; Geitler, 1965). However, the present
findings may indicate a possible direction for further research.
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(Received 13 January 1969—Revised 16 May 1969)
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Fig. 4. P. coccineus, highly endopolyploid suspensor cell with polytene chromosomes.
Note the size difference between this and the cells and nuclei of the integument and
endosperm, which are respectively diploid and triploid. Original relationships of cells
altered by the squash technique. Dark-field, x 160.
Fig. 5. P. coccineus, vacuolated nucleolar lobes of a highly endopolyploid suspensor cell
nucleus, at 27 °C. Arrows indicate a divided satellite. Phase contrast, x 1600.
Polytene chromosomes in Phaseolus
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Figs. 6-11. P. coccineus, suspensor. Phase contrast, x 1600.
Fig. 6. Nucleolus organizer of polytene chromosome I, splitting up at 27°/22 °C.
Arrow indicates the centromere.
Fig. 7. Satellite of polytene chromosome I, ramified. 27°/22 °C.
Fig. 8. Nucleolus organizer of polytene chromosome I, condensed. i2°/8 °C. The
arrow indicates the centromere.
Fig. 9. Satellite of polytene chromosome I, compact. i2°/8 °C.
Fig. 10. Small additional nucleolus (arrow) originating on a heterochromatic filament
starting from a polytene chromosome. 27°/22 °C.
Fig. 11. Additional nucleolus originating from the distal band of a polytene chromosome. 27°/22 °C. Note the chromatic areas within the vacuoles (arrows).
Polytene chromosomes in Phaseolus
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Figs. 12-17. P- vulgaris, suspensor. Phase-contrast, x 1600.
Fig. 12. Part of the condensed secondary constriction of a polytene chromosome
visible within an intranucleolar vacuole (arrow). i2°/8 °C.
Fig. 13. Loosened threads of the secondary constriction with a fuzzy appearance,
visible within an intranucleolar vacuole. 27°/22 CC.
Fig. 14. Gigantic vacuole of a nucleolar lobe (indicated by arrows). The 'puffed'
secondary constriction is visible as dispersed chromatin within the vacuole. Following
a temperature shock of 35 °C.
Fig. 15. Part of a condensed secondary constriction with a banded appearance. Following a temperature shock of 50 CC.
Fig. 16. Additional, free nucleolus with an extrusion. 27°/22 °C.
Fig. 17. Main nucleolus of a low endopolyploid cell, with many nucleolar extrusions.
Following a temperature shock of 35 °C.
Polytene chromosomes in Phaseolus
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Figs. 18-26. P. coccineus, suspensor. Phase-contrast, except Fig. 21. x 1200, except
Fig. 22 which is x 240x3.
Fig. 18. Part of a nucleus with polytene chromosomes in the lampbrush state (arrows).
Two hours after temperature increase from 150 to 30 °C.
Fig. 19. Lampbrush polytene chromosome, squashed out from the nucleus; 2 h after
temperature increase from 15° to 30 °C.
Fig. 20. Late lampbrush state of a polytene chromosome in which most of the loops
are retracted; 4 h after temperature increase from 15° to 30 °C.
Fig. 21. Toluidine blue-stained polytene chromosome in the lampbrush state. The
loops appear as a veil around the chromosome (arrow). The cell was hydrolysed for
30 min before staining.
Fig. 22. Higher enlargement of a single loop (arrow).
Fig. 23. Degenerating polytene chromosome, to demonstrate the differences from the
looped state. Note the longitudinal disintegration.
Fig. 24. Droplets, occurring above lampbrush polytene chromosomes.
Fig. 25. Filling up of networks formed by loops, to form nucleolus-like bodies
(arrows).
Fig. 26. Empty networks (arrows), and a disengaged nucleolus-like body (double
arrow).
Polytene chromosomes in Phaseolus
107