J. Cell Set. a8, 87-105 (1977)
Printed in Great Britain © Company of Biologists Limited igy7
87
NUCLEAR CHANGES ASSOCIATED WITH
CELLULAR DEDIFFERENTIATION IN
PEA ROOT CORTICAL CELLS CULTURED
IN VITRO
LEWIS J. FELDMAN* AND JOHN G. TORREYf
Department of Biology, Harvard University, Cambridge, Massachusetts 02138, U.S.A.
SUMMARY
Root cortical tissues explanted from seedling roots of the garden pea, Pimm sativiun L.,
cv. Little Marvel cultured in sterile medium containing indoleacetic acid and kinetin were
fixed, sectioned and studied with the electron microscope. Nuclear changes over 60 h of culture
were examined during the events of dedifferentiation of mature parenchyma cells into subdivided newly meristematic derivatives. Table 1 summarizes the events which are evident in
7 population classes designated I—VII. The initially small, round nucleus with a single,
unvacuolated nucleolus and highly condensed and disperse chromatin showed marked volume
increase, nucleolar enlargement of almost 20-fold, development of diffuse and then clumped
chromatin and then subdivision of the nucleoli into a number (5-10) of small nucleoli
immediately preceding cell division. Mitoses involved cells with diplochromosomes and daughter
nuclei with doubled chromosome number, either 471 or 8n. Subdivided cortical cells showed
an increased ratio of nucleus to cytoplasm although the nuclei were reduced in volume to that
of the original nuclear population. Nucleoli in subdivided cells were small and unvacuolated and
chromatin changed from an initially condensed state through a diffuse and disperse condition
to a condensed form. Cortical cells divided at least twice in rapid succession before initiating
new events leading to redifferentiation. Nuclear changes in dedifferentiation in pea were comparable in many respects to the similar process studied in animal systems including eye lens
regeneration, limb regeneration and activation of division in blood lymphocytes. The cytological
changes in nuclear structure could be correlated with documented changes in DNA, RNA and
protein levels in these systems. By 60 h cells presumed to be ready for redifferentiation and
cell specialization were observed in the subdivided population but structural evidence for
commitment to a new course of cytodifferentiation was not obtained.
INTRODUCTION
The early events associated with the differentiation of a totipotent cell into a particular cell type are only vaguely understood. In part, this problem arises because of
the difficulty in specifying precisely when a cell is set on the course of cytodifferentiation. For this reason, it is often impossible to determine exactly when and
to which cytological and biochemical events a particular cell owes its origin. In
investigations of cytodifferentiation therefore, clear advantage often can be gained by
• Mailing address: Department of Botany, University of California, Berkeley, California,
94720, U.S.A.
f Permanent address: Cabot Foundation, Harvard University, Petersham, Massachusetts,
01366, U.S.A. (Please send reprint requests to this address.)
88
L. J. Feldman and J. G. Torrey
employing a system in which one induces an already determined cell type to differentiate into an entirely different cell product.
In both plants and animals, examples exist in which an already determined cell can
be induced experimentally to dedifferentiate and subsequently to redifferentiate into
a new cell type. During the course of this cytodifferentiation numerous cytological
and metabolic processes must occur. Included in these processes are the loss of the
characteristics which distinguish a particular cell type, and the assumption of processes
(cytological, biochemical) which mark the cell as ^differentiating into a new cell type.
Thus, one is dealing with 2 distinct, but not necessarily separable events, the reversion
of a mature differentiated cell to a meristematic state and the reactivation of the
dedifferentiated cell into an entirely new course of cytodifferentiation.
In plants one of the most carefully studied systems employs cultured pea root
cortical explant tissue. In this experimental system a large proportion of the population of already mature, differentiated cortical cells is induced to dedifferentiate and
subsequently redifferentiate into tracheary elements. The trigger for these processes
are hormones: i.e., auxin and cytokinin, which, when present in the tissue culture
medium, initiate a set of events culminating in the formation of tracheary elements
from subdivided cortical cells.
Some of the biochemical and cytological processes associated with the differentiation of tracheary elements in pea root cortical explant tissue have been documented
in recent papers with special emphasis on DNA synthesis and related nuclear events
(Torrey & Fosket, 1970; Libbenga & Torrey, 1973: Phillips & Torrey, 1973). At the
light-microscope level the time course of this cytodifferentiation involves a number of
distinct phases. The cortical cells with interphase nuclei at 2C and 4 C levels of DNA,
initially all diploid, begin DNA synthesis 24 h after subculture. As a result of this
synthesis, several discrete populations, most of which are polyploid, are produced
prior to the first observed mitoses. The first cells to divide are usually tetraploid,
although thereafter the level of ploidy may vary in populations of dividing cells, i.e.,
4« and 8n. Five to seven days after culturing the explant, secondary cell walls
characteristic of tracheary elements are observed in derivatives of recently divided
cortical cells as the first morphological evidence of redifferentiation.
At the electron-microscope level Bowes & Torrey (1976) examined early ultraStructural changes in these cell populations, including in particular, changes in the cell
walls. In explants as old as 72 h in culture they were unable to observe any direct
evidence that divided cortical cells were yet committed to differentiate into tracheary
elements.
In the work presented here, ultrastructural observations were extended to include
a detailed study of nuclear morphology. The distinct nuclear populations as revealed
by the electron microscope were evaluated in the light of autoradiographic and microspectrophotometric evidence concerning the onset and magnitude of DNA synthesis
in differentiating cortical cells and in studies correlating ultrastructural images of
nuclei with those at the light-microscope level specially stained to show nucleolar
structure.
Nuclear changes in cultured pea root cells
89
MATERIALS AND METHODS
Seed of Pisum sativum, cv. Little Marvel, were surface sterilized in commercial Chlorox
(sodium hypochlorite solution), rinsed 3 times in sterile distilled water and allowed to imbibe in
distilled water in the dark for 16 h. The seeds were then placed aseptically with the radicle end
up in Petri plates containing o-8 % agar dissolved in distilled water, and germinated in the dark.
From roots 15-25 mm in length segments 1 mm thick were cut 10—11 mm proximal to the
root tip. The vascular cylinder was removed by the technique described by Libbenga & Torrey
(1973) and the cortical explants were transferred aseptically to Petri plates containing SzM
medium supplemented with 10 mg/1. kinetin and o-8 % agar. The explants were cultured in
the dark at 25 °C.
Electron microscopy
Segments were fixed in either 3 % glutaraldehyde for 25 h at room temperature or at
o °C in 10 % acrolein for 48 h, washed and subsequently postfixed in 2 % osmium tetroxide for
2 h at room temperature or for 24 h at o CC; all solutions were prepared with 0025 M sodium
phosphate buffer, pH 6-8. Segments were dehydrated initially through a graded acetone series
and then through a graded absolute acetone-propylene oxide mixture and embedded in EponAraldite (Phillips & Torrey, 1974). Sections were cut on a Reichert ultramicrotome and were
stained in 2 % uranyl acetate for 30 min and in lead citrate for 4 min (Reynolds, 1963), then
dried and examined with a Philips 300 microscope.
Symbols used on electron micrographs
c
CC
cp
cw
cy
er
f
8
is
chromatin
condensed chromatin
cell plate
cell wall
cytoplasm
endoplasmic reticulum
flbrillar
granular
intercellular space
/
m
new
nm
no
nu
nv
V
lacunae
mitochondrion
new cell wall
nuclear membrane
nucleolus organizer
nucleolus
nucleolar vacuole
vacuole
Calculations of nuclear and nucleolar volumes from squashes prepared for light mict oscopy
Segments were fixed for 24-48 h in Craf II, washed overnight, hydrolysed in I N HC1 at
60 °C for 8 min, stained in Schiff reagent in the dark for 35 h and rinsed 3 times, 10 min each,
in SO2 water. The explant tissue was then rinsed and treated for 3 h at room temperature with
°'3 % pectinase in 02 M sodium acetate buffer, pH 45. Segments were then squashed on
albumin-coated slides, heated slightly and the coverslips floated off in 50% ethanol. Coverslips,
to which the squashed segments were affixed, were rinsed briefly in water, air dried, and
counterstained in 015 % methylene blue (aqueous) so that nuclear and nucleolar detail could
be distinguished. In calculating nuclear and nucleolar volumes, nuclei and nucleoli were taken
as spherical, their volumes being calculated as 4/3 nrs. The radius, r, was obtained by measuring
2 diameters at right angles and halving the average value, since neither the nucleus nor nucleolus
was perfectly spherical. The values obtained are therefore somewhat approximate.
Time
Populain
tion culture,
no.
h
,
D*
r
vt
A
Nucleus
I.
5'1
2.5
Large,
appressed
to wall
Nucleus to
centre of
cell. Large,
diffuse, ovoid
+
4' 1
Still flat,
appressed to
wall. Doubled
size
2
<
Small
elongate,
appreased to
cell wall
D+
354
434
299
17
vt
A
Diameter, pm.
7 7
A
,-.
t
Chromatin more diffuse;
granular and fibrillar
zones intermingled.
Nucleolus organizer
evident
Volume, pma.
-+
4C
3;16,17
I, 2; I5
8C andlor
Chromatin clumped and
peripheral; nucleolus
I 6C
with large outer granular
and smaller inner fibrillar
zones, with vacuoles.
Nucleolus organizer associated with periphery of
the nucleolus
5-8;
21-23
Some
4 ; 18-20
cases
involving
endoreduplication
DNA
synthesis
begins
24 h
Chromatin highly con2C
densed and disperse in
nucleus. Nucleolus shows
both granular and fibrillar
zones. Nucleolus organizer
evident
Distinctive
ultrastructural
appearance
Postulated
D N A amounts
(based on
data from
Libbenga & Illustrated
Torrey,
in figure
1973)
no.
Present,
Chromatin clumped and
large and peripheral ; nucleolus
central
large and vacuolated. N o
nucleolus organizer seen
Small or
absent;
numerous
lacunae
Small or
absent;
small
lacunae
Nuclear
vacuoles
From single Multiple
to multiple
and small
(5-10) to
disappearing
Single, large
Single
enlarged
Single,
round,
small
Nucleolus
-
Summary of nuclem events during culture
Dimensions and state of
Table
7
9
'
..
8
5
g
m
%
4
P
o
\O
Niulem changes in cultured pea root cells
92
L. J. Feldman and J. G. Torrey
RESULTS
Electron microscopy of the nucleus
Nuclear morphology was examined at time o immediately after explanting and at
42 and 60 h after explanting. At 42 h the population as a whole showed peak
[3H]thymidine incorporation activity (in parallel studies, 60% of the cortical cell
nuclei showed incorporation after a 6-h-label period); at 60 h the population showed
a peak mitotic index (~ 6-5 % of the cells were in mitosis. See also fig. 7 in Phillips &
Torrey, 1973). For clarity in discussions seven populations of cells were designated,
related to the time in culture and to a presumed sequence of change. The observations
on each population are described below and summarized in Table 1.
Table 2. Nuclear dimensions in the 7 cortical populations of Table 1 determined from
light-microscopic measurements of fixed squash preparations stained with Schiff's reagent
counterstained with methylene blue
Time
Volume of nucleus, /im3
Radius of nucleus, /tin
in
Popula- culture,
h
tion
,
High
Low
I
II
III
IV
0
6-6
4-2
42
io-o
79
42
60
125
129
V
VI
VII
60
60
60
108
II-2
Average
^
5-i
94
"•5
124
A
1
High
Low
1200
4119
8200
9000
312
542
2065
5280
5880
3420
6370
8100
310
1000
1000
1500
1500
Average
First mitosis; 2 nuclei
5-8
79
8-3
4-2
62
62
5i
7-1
7-1
815
2065
2420
556
Each sample is based on measurements of 5-6 nuclei.
Time o (population I nuclei)
In the cultured explant at time o, the cortical cells are large, averaging 40 x ioo/tm
and possessing an extensive vacuole and a thin parietal cytoplasm (cf. Bowes &
Torrey, 1976). The nuclei are small, flattened and elongate, appressed to the cell wall
with an average diameter of about 5 /tm (Table 2). The nuclear membrane is smooth
and unlobed. As noted by Lafontaine & Lord (1974) and by Chaly & Setterfield
(1975), nuclei in cortical cells of pea are composed of an extensive network of highly
condensed chromatin* often attached to the nuclear membrane and intermingled with
less-dense regions in the nucleoplasm. Each nucleus contains a single, round nucleolus,
1-6-2-0/(m in diameter, only rarely associated with the chromatin (Figs. 1, 15). The
granular zone of the nucleolus is peripheral and fairly extensive, comprising approximately one-half of the nucleolar volume. Internal and surrounded by the granular
zone is a distinct and large fibrillar zone, occupying usually one third to one half of
• Because of problems of definition (see Chaly & Setterfield, 1975), the terms heterochromatin and euchromatin will not be used.
Nuclear changes in cultured pea root cells
93
the nucleolar volume (Fig. 2). Small lacunae are occasionally noted within the fibrillar
zone. Nucleolar vacuoles, that is, lighter-staining regions within the granular material,
are lacking. The nucleolar organizer region is usually evident, most often near the
periphery of the nucleolus.
In their measurements of relative DNA amounts in nuclei of time o pea root
cortical cells, based on total fluorescence of DNA-specific dye in whole nuclei studied
in sectioned material, Libbenga & Torrey (1973) found 2 distinct classes of nuclei
with respect to their DNA content. One class was interpreted as 2C and the other as
4C. No nuclear dimensions were measured in these experiments. We have used the
squash method described in Methods to prepare time o cortical cells to try to distinguish 2 nuclear classes with respect to nuclear radius or volume. Measurements of
over one hundred nuclei gave a single bell-shaped curve with a continuous range of
nuclear radii varying from 3-6 to \2-o fim, average 7-6/jm. This value, which represents one class discernible by radial dimensions, agrees well with the dimensions
derived from the much smaller samples given in Table 2. It must be presumed therefore that DNA values ranging from 2C to 4C are subsumed within this single volume
class.
Nuclei in explants cultured for 42 h {populations I-III)
In explants cultured for 42 h 3 distinct types of nuclei, designated populations I,
II and III were observed. In most respects nuclei of population I resembled the time o
nucleus and are assumed to be unresponsive to culture. Nuclei of population II have
enlarged considerably with an increase in nuclear and nucleolar diameters approximately twice that observed in the time o nuclei (see Figs. 16, 17 and Tables 1 and 2).
Such nuclei are more lobed, but still adjacent to the cell wall. Chromatin is dispersed
and no longer found in dense clumps as at time o. Occasional connexions are observed
between the nucleolus and the chromatin. A peripheral, not very compact granular
zone is present in the nucleoli (Fig. 3). Internally, fibrillar and granular materials have
become intermingled. An increase in the number of small lacunae, almost exclusively
associated with the fibrillar zone are observed in population II (Fig. 3). Occasional
small vacuoles are noted within the granular zone and nucleolar organizer regions are
noted frequently, usually in a somewhat more interior position than in the time o nuclei.
Nuclei of population III are readily distinguished by the presence of a large
nucleolar vacuole (Figs. 4, 18-20). The nuclei are larger than those of population II
(Table 2) and less regular in shape. The chromatin, occasionally seen as large dense
clumps, is peripheral in distribution, sometimes attached to the nuclear membrane,
but is rarely in direct contact with the nucleolus. Nucleoli are round to slightly ovoid
with extensive granular and fibrillar zones. Occasionally the granular zone extends as
a continuous band from the exterior to the interior of the nucleolus. Small lacunae are
distributed throughout the fibrillar material. Within the central vacuole, which comprises approximately one third to one half of the diameter of the nucleolus, are
numerous small granular particles, sometimes appearing connected in chains. Nucleolar organizer regions are not observed in nucleoli containing a large central vacuole.
L. J. Feldman and J. G. Torrey
A7/7if
Fig. i. Nucleus at the time of explanting (population I). The chromatin is highly
condensed. The nucleolus is relatively small, x 7850.
Fig. 2. Nucleolus of a population I nucleus. Note that there is little intermingling
between peripheral granular and interior fibrillar zones, x 30200.
Fig. 3. Nucleus 42 h after explanting (population II). Note the numerous lacunae in
the nucleolus and the highly intermingled nature of the granular and fibrillar zones.
X6170.
Fig. 4. Nucleus 42 h after explanting (population III). Note the well developed nucleolar vacuole surrounded by lacunae and the highly intermingled nature of the fibrillar
and granular zones in the nucleolus. x 9050.
Nuclear changes in cultured pea root cells
. nm
95
v
nm
8
Fig. 5. Nucleolus of an undivided cell 60 h after explanting (population IV). Note
the greatly enlarged nucleolus and nucleolar vacuole. The chromatin is typically
clumped and peripheral. X5150.
Fig. 6. Enlarged portion of Fig. 5 showing detail of the nucleolus organizer. Note the
extensive peripheral granular zone of the nucleolus. x 27650.
Fig. 7. Nucleus of an undivided cell, 60 h after explanting. Note the multiple nucleoli,
each with a halo of granular particles and a compact central fibrillar zone. The
chromatin is more uniform in distribution; compare with Fig. 5. This nucleus is
probably preparing for its first mitosis, x 6700.
Fig. 8. Enlarged portion of the nucleus of an undivided cell. Arrows point to possible
remnants of the nucleoli. x 11 200.
96
L. J. Feldman and J. G. Torrey
Nuclei in explants cultured for 60 h [populations IV-VII)
This tissue is comprised of several discrete populations of cells including some
fitting the descriptions of populations I—III and additional cell types, designated
populations IV-VII. Included are cells which have not yet divided, but with marked
nuclear changes, cells which have divided once, and cells which have divided twice,
subdividing the original cortical cell into 4 smaller cells. By comparing the thickness of
cell walls, it is possible to determine how many divisions preceded the formation of
any given cell as was shown by Bowes & Torrey (1976).
Within the populations of undivided cells which responded to the culture treatment, population IV is a broad category which includes cells with a single large
nucleolus, cells with multiple small nucleoli, and cells about to enter cell division with
accompanying disappearance of the nucleolus. Nuclei of some of these cells have
reached their greatest measured size (population IV, Table 2), increasing 15-16 times
in nuclear volume as compared to the time o nuclei (Figs. 21, 23). The nuclei are no
longer appressed to the cell wall, but rather are located in a more central position
within the cell (Fig. 5). The chromatin is seen as discrete, dark-staining masses
peripheral in distribution, often in contact with the slightly lobed and undulate
nuclear membrane. The enlarged nucleolus consists primarily of numerous zones of
internal fibrillar material bounded by a peripheral granular zone. Within the granular
zone, which comprises approximately two-thirds of the nucleolus, are several large
unequal-sized vacuoles. Eachvacuole contains dark-staining granular material (Fig. 6).
Numerous small lacunae are evident within the fibrillar zones. Nucleolar organizers
are evident as spherical bodies in direct contact with the nucleolus proper (Figs. 5, 6).
Material comprising the nuclear organizer is more fibrillar in nature when compared
to the distinctly granular zones around the large nucleolar vacuoles and appears to be
permeated by small channels.
In the undivided, responsive cell at a slightly later stage but still in population IV
the large nucleolus has dissolved into 5-10 small nucleoli, with rough, irregular
edges, and apparently composed mainly of fibrillar material, bounded by a halo of
granular particles (Figs. 7, 22, 23). The nucleus with its nuclear membrane still intact
becomes slightly ovoid and more or less lobed. The chromatin condenses into scattered,
dark-staining patches and is no longer peripheral in distribution. In some cells, after
the nucleoli have completely disappeared, small circular bodies can be observed
(Fig. 8), which may be remains of the nucleoli.
In Fig. 13 is seen a cortical cell in early prophase of its first division. The nuclear
envelope has broken but is still apparent at the periphery of the nuclear area. The
condensed chromosomes are cut in section and show a striking paired arrangement,
probably demonstrating their diplochromosomal nature. No specialized structures
related to this endoreduplicative origin are evident other than the pairing. At
metaphase, these chromosomes will separate, giving rise to the tetraploid chromosome
number. Here, about 25 of the total number of 28 chromosomes are seen in section.
Nuclear changes in cultured pea root cells
Fig. 9. Nucleus of a once-divided cell, 60 h after explanting (population V). Note the
enlarged nucleolus, and highly condensed, discrete clumps of chromatin. x 3520.
Fig. 10. Sister nuclei, the result of the first division of a cortical cell (population V).
Note the highly compact nature of the nucleoli and the absence of vacuoles or lacunae.
X7650.
Fig. 11. One of the daughter nuclei of a once-divided cortical cell (population VI).
Compare this nucleus with those in Fig. 10. x 4670.
Fig. 12. Daughter nucleus of a once-divided cortical cell. It may be preparing to
undergo a second mitosis (population VII). Compare with Fig. 7. x 5900.
L. J. Feldman and J. G. Torrey
I
nm
13
cw
cw
V
14
Fig. 13. Metaphase in a nucleus undergoing its first mitosis. This section cuts portions
of about 25 chromosomes, x 6050.
Fig. 14. Sister nuclei typical of a subdivided cortical cell after completion of the new
cell wall. The condensed chromatin suggests that these nuclei are relatively inactive,
x 4670.
Cells at 60 h which Itave divided [populations V-VII)
In the once-divided cells (population V) the nucleus is more or less circular in
section and remains centrally located in the cell adjacent to the newly formed cell wall
(Figs. 9, 10). The nuclear diameter and volume may have been reduced to that of the
original size seen in population I (Table 2) or may be intermediate in size. In such
nuclei the chromatin is distributed at the periphery of the nuclear envelope as dark,
m
Nuclear changes in cultured pea root cells
26
Figs. 15-26. Squashes of whole nuclei from explants cultured 48 h, stained with the
Feulgen method and methylene blue. All x 1450. In Figs. 22 and 23 note the multiple
number of nucleoli per nucleus. The 2 sister nuclei in Fig. 26 (arrows) are products of
either the first or second mitosis of a polyploid cortical cell.
i oo
L. J. Feldman and J. G. Torrey
highly condensed masses. Occasional contact is observed between the chromatin and
the nuclear membrane and/or peripheral zones of the nucleolus. The nucleoli,
generally one per nucleus, are lobed or somewhat irregular in shape and may occupy
a relatively large portion of the nucleus. A peripheral granular zone is usually observed,
occasionally completely encircling the more internal fibrillar matrix. In the interior
of the nucleoli the granular and fibrillar zones may or may not be highly intermingled.
Nucleolar lacunae are small and numerous and often contain a lighter-staining material
which may be chromatin.
Such cells may enter promptly into cell division again so that in 6o-h samples,
cortical cells may have divided once or twice (cf. figs. 11,13 m Bowes & Torrey, 1976).
Nuclei immediately after such divisions give the appearance described as population V.
In Fig. 10 are shown 2 nuclei with the new cell plate just forming between them. Each
contains a single, non-vacuolated nucleolus and chromatin-rich nucleoplasm. Although
it is difficult to be certain from static sampling (as compared to time-lapse cinemicrography, for example), it is probable that such nuclei in just-divided cells undergo
the sequence described in Table 2, going on to form cells of populations VI and VII.
The nuclei undergo a diameter and volume increase (population VI) and are often
characterized by the presence of a single large nucleolus with a large central vacuole
(Figs. 11, 24, 25). Chromatin becomes more diffuse and disperse. Nucleoli appear
composed largely of fibrillar material. The new cell wall is well formed and the ratio
of nucleus to cytoplasmic volume is relatively high.
Immediately prior to the next cell division, these new meristematic cells are
characterized by the presence of multiple nucleoli (Fig. 12), a phase which appears to
precede nuclear division, repeating the sequence observed in populations III and IV
prior to the first division. If the cells do not divide further, they arrest at the stage VI
condition with nuclei containing only a single large nucleolus with a large vacuole
(Fig. 14). Fig. 26 may be compared to the cells from a first division (Fig. 10) or may
result from a second division, a fact not determinable from squash preparations.
Fig. 14 represents subdivided cortical cells after either the first or second division in
which the nuclei give the appearance of relative inactivity, i.e., condensed chromatin
and large vacuolated nucleoli. Such cells are reminiscent of population I cells but may
be cells prepared now to redifferentiate. Evidence for such a sequence remains to be
obtained.
DISCUSSION
Cortical cells before division
In undivided cortical cells, a pronounced enlargement of the nucleus and its
nucleolus is one of the first and most obvious indications that a cell is responsive to
hormonal stimulus. Phillips & Torrey (1973) showed that approximately 60% of the
initial explant population responds to excision and culture by undergoing DNA
synthesis. This observation, which has been confirmed in detail repeatedly, corresponds well with what is noted at the ultrastructural level. In the nuclei of enlarging,
undivided cells the abundant, condensed chromatin of a typical time o nucleus
Nuclear changes in cultured pea root cells
101
disappears with a concomitant increase in the regions of diffuse chromatin. Various
workers (Tokuyasu, Madden & Zeldis, 1968) have shown that it is within the loosened,
highly dispersed state of chromatin that the most active DNA synthesis occurs. In
several plant systems (Deltour & Bronchart, 1971; Jordan & Chapman, 1971, 1973;
Lafontaine & Lord, 1974) as well as in a variety of animal systems (Hay, 1959;
Tokuyasu et al. 1968) the decondensation of the chromatin is associated with early
and rapid synthesis of DNA. Thus, it appears likely that renewed DNA synthesis in
cortical cells is at least in part a consequence of the dispersal of the chromatin, as has
already been suggested in other reactivated tissues (Dumont & Yamada, 1972; Harris,
1967).
Since it is known from [3H]thymidine incorporation data that DNA synthesis in
cortical explant cells does not commence until approximately 24 h after subculturing
(Phillips & Torrey, 1973), any modification of the chromatin reticulum which occurs
from o to 24 h must not take place during the S period. This suggests that mature
cortical cells are normally stabilized in the Gx period of the cell cycle. Similar conclusions have been reached with regard to adult iris epithelial cells involved in
Wolffian lens regeneration (Reese, Puccia & Yamada, 1969).
In the Wolffian lens regenerating system, marked RNA synthesis precedes activation
of DNA synthesis. In the pea cortical explant system we have not yet examined in
detail the occurrence of biosynthetic events which may precede DNA synthesis.
Shininger & Polley (1977) reported that pea root cortical explants showed a 2- to 4fold enhancement of the rate of RNA synthesis compared to controls lacking hormone.
Stimulation of RNA synthesis could be detected as early as 9 h after explanting. In
the same system, Simpson & Torrey (1977) showed that in the presence of hormones,
protein accumulation began after 2 days, with a steady increase through day 4. It was
inferred that during the first 2 days, protein degradation occurred, thereafter net
synthesis of protein was apparent, leading to cell division. Thus, in pea, preliminary
work indicates that protein synthesis is greatly enhanced subsequent to explant
culture, suggesting either a more accelerated activity of, or a marked increase in the
numbers of ribosomes. Observations on the ultrastructure of the nucleolus, where
precursors of ribosomal RNA are believed synthesized (Brown & Gurdon, 1964;
Perry, 1967; Birnstiel, 1967), support the latter proposal, that is, that increased
rRNA synthesis occurs in the activated cells.
In responsive cortical cells in culture, nucleoli show an increase in the proportion
as well as in the absolute amount of granular material, a breaking up of the extensive,
compact fibrillar region into numerous smaller packets, and the formation of a central
enlarged vacuole, itself bounded by a granular zone. In parallel to these ultrastructural
changes either the nucleolar volume increases or the number of nucleoli per cell
increases and the frequency of cells whose nucleus contains multiple nucleoli increases.
These changes were also noted by Fowke & Setterfield (1968) in cultured cells of
Jerusalem artichoke and by GifFord & Nitsch (1969) in tobacco pith cells. Each of these
nucleolar modifications is believed associated with an increased capacity for synthesis
of precursors of rRNA (Rose, 1974). For example, evidence suggests that, in nucleoli
actively synthesizing precursors to rRNA, the granular and fibrillar zones are highly
102
L. J. Feldman and J. G. Torrey
intermingled (Hyde, Sankaranarayanan & Birnstiel, 1965; Rose, 1974) and that these
2 zones become segregated in relatively inactive nucleoli. In other systems (Johnson,
1969) the presence of a large central vacuole was correlated with the incorporation of
relatively more [3H]uridine as compared to nucleoli lacking vacuoles. In the Tetrahymena nucleolar system Nilsson & Leick (1970) suggested that the dissociation of
large nucleoli into more numerous smaller units may serve as an efficient mechanism
for synthesizing precursors of rRNA. Thus, Hyde (1967), Miller & Beatty (1969) and
many others have considered that nucleolar structure is a valid indicator of nucleolar
activity in rRNA synthesis.
As the cortical cell prepares to undergo its first mitosis after the S period is completed (population IV), the large, single nucleolus is replaced by 5-10 small nucleoli
in which separation between the granular and fibrillar regions becomes more or less
indistinct. Such nucleoli are probably no longer active in synthesis of rRNA precursors. Nuclei with multiple nucleoli show chromatin in the form of large compact
masses peripherally distributed. Then the nucleoli disappear and the cell enters
mitosis and cytokinesis, forming 2 new cells.
Cortical cells after division
From the experiments of Phillips & Torrey (1973) and Libbenga & Torrey (1973)
we know that the first divisions occur in cortical cells whose nuclei contain DNA
amounts higher than the normal diploid populations. Frequently such cells are
tetraploid at the first mitosis or may even be octaploid. These cells may undergo
another round of DNA synthesis and then divide a second time at the same ploidy
level within 60 h of culture.
At the ultrastructural level, there is no clear indication of the ploidy level of these
cells. From Table 2 one might be inclined to relate the marked increase in nuclear
volume observed with the light microscope in population IV nuclei to the known
changes in DNA values. But there appear to be no reliable volume changes which can
be used as a measure of the ploidy level since after the first division, nuclear volumes
diminish to essentially time o dimensions. Efforts to discern distinctive nuclear changes
associated with endoreduplication failed although evidence for polyploid chromosome
number was found.
Each daughter cell of the first cortical division usually contains a relatively small
nucleus in which the chromatin is initially condensed, then becomes disperse. The
single nucleolus is large relative to the nuclear size and irregular in shape. Fibrillar
material is present but the granular zone is not easily distinguished. Observations of
the ultrastructure of these nucleoli suggests they are not very active in rRNA
synthesis. However, the ratio of nucleus to cytoplasm has increased and the total
cytoplasm per cell has increased so that the cells take on the appearance of a more
meristematic state (see figs. 9-14 in Bowes & Torrey, 1976).
The nature of dedtfferentiation
From several animal systems in which an already determined cell is induced to
differentiate into a new cell type (e.g., iris lens regeneration, Dumont & Yamada,
Nuclear changes in cultured pea root cells
103
1972; limb regeneration, Jeanny & Gontch-Aroff, 1974; activation of peripheral blood
lymphocytes, Tokuyasu et al. 1968) investigators have shown that the initial steps in
dedifferentiation involve the following events: the decondensation and dispersal of
chromatin (Dumont & Yamada, 1972); the enlargement of nucleoli and an associated
increase in the complexity and distribution of the fibrillar and granular zones (Hay,
1959); an increase in RNA synthesis, usually preceding DNA synthesis (Hay &
Fischman, 1961); an increase in DNA synthesis (Killander & Rigler, 1965); the
acquisition of nuclear and cytoplasmic characteristics considered necessary for division
(Jeanny, 1973); the activation of other biochemical processes (Darzynkiewicz, Bolund
& Ringertz, 1969; Killander & Rigler, 1965).
According to Tokuyasu et al. (1968), in lymphocyte activation, RNA synthesis
begins about 24 h after initial stimulation. Hay & Fischman (1961) reported that
intracellular protein synthesis occurs early in the dedifferentiating cell in limb
regeneration. Jeanny (1973) suggested that in limb regeneration in Desmognathus an
increase of cytoplasmic contents per cell is a necessary step in dedifferentiation leading
to mitosis.
Dedifferentiation in pea root cortical cells shows striking parallels to this sequence
of events. DNA synthesis begins approximately 24 h after subculturing explants
(Phillips & Torrey, 1973). RNA synthesis occurs as early as 9 h after explanting and
then increases thereafter (Shininger & Polley, 1977). This timing would coincide well
with the dramatic increase in nucleolar size and internal complexity observed in this
study which is suggestive of rRNA synthesis.
Bowes & Torrey (1976) noted an increase in the number of organelles, especially of
free ribosomes and rough ER associated with the onset of DNA synthesis and
thereafter. Simpson & Torrey (1977) reported the increase in net protein synthesis
beginning at about 48 h and continuing into the fourth day in culture. Such synthesis
is presumed to be associated with the general increase in cytoplasmic density and
complexity observed in these cells as they revert to the meristematic state.
Evidence of redifferentiation
In the literature on animal systems the events most often considered associated with
redifferentiation of a new cell type are mitosis (Yamada & Roesel, 1971) followed by
the cessation of DNA synthesis (Dumont & Yamada, 1972), followed by the production
of cell-specific products. In cells which have divided, nuclear chromatin becomes
condensed, symptomatic of the cessation of further DNA synthesis. In such cells, the
redifferentiation process is suggested to have already begun (Dumont & Yamada, 1972).
In the pea root cortical cell system the relationship between DNA synthesis and
redifFerentiation is complicated by the occurrence in many cells of DNA synthesis by
endoreduplication. The evidence is strong that tracheary element formation occurs in
subdivided endoreduplicated cortical cells. The role of polyploidization in this system
is unclear since tracheary elements may differentiate independent of specific DNA
levels (Phillips & Torrey, 1974). It is possible that polyploidization is not directly
related to tracheary element differentiation, but rather represents a mechanism for
enhancing rRNA synthesis, a possibility quite consistent with the observation by
104
L. J. Feldman and J. G. Torrey
Bowes & Torrey (1976) of the marked increase in both free and polyribosomes in
cells which are undergoing endoreduplication. The present study documents the
changes which occur in nuclei of mature cortical cells induced to undergo dedifferentiation by hormonal stimuli. Further studies will be necessary to gain insight into
the determinative events leading to ^differentiation.
The authors are indebted to the National Science Foundation for partial support of this
research under research grant BMS 74-20563.
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(Received 18 March 1977)