Download Nuclear DNA endoreduplication during petal

Journal of Experimental Botany, Vol. 53, No. 371, pp. 1017–1023, May 2002
Nuclear DNA endoreduplication during
petal development in cabbage: relationship between
ploidy levels and cell size
Nobuhiro Kudo1 and Yasuo Kimura
Division of Plant Biotechnology, Gunma Horticultural Experiment Station, 493 Nishi-Obokata,
Sawa-Azuma, Gunma 379-2224, Japan
Received 5 July 2001; Accepted 3 December 2001
Abstract
The development of cabbage petals comprises two
distinct phases: a cell division phase and a consecutive phase of cell expansion until the onset of opening.
In this study, cytological changes characterizing
the two phases of petal development were analysed.
First, the mitotic activity and the surface area of epidermal cells during petal development were investigated. The DNA content of isolated nuclei from the
different stages of petal tissues was determined by
flow cytometric analysis. The results show that cell
differentiation, leading to expanded cells, is characterized by endoreduplication. In the proximal part of
the petal, after cell division arrest, differentiation frequently involves endoreduplication and cell enlargement. By contrast, normal diploid nuclei remained in
the distal part of the lamina in the mature petal. It
is suggested that the developmental programmes of
the cabbage petal may be a trigger for the initiation of
endoreduplication. Correlation between ploidy levels
and cell size is also discussed.
Key words: Brassica oleracea L., endopolyploidy, endoreduplication, flow cytometry, petal development.
Introduction
Endoreduplication is an alternative form of cell cycle
that occurs in a wide variety of organisms (Nagl, 1976;
Barlow, 1978). During endoreduplication, nuclear DNA is
replicated without mitosis or cytokinesis, resulting in cells
with DNA content much greater than 2C (C is the haploid
DNA content). The occurrence of endoreduplication is
very frequent in arthropods. Endoreduplicated chromosomes (polytene chromosomes) is a nuclear differentiation
1
mechanism in larval and adult Diptera (Pearson, 1974). A
variety of cell types in Drosophila larval tissues undergo
endoreduplication events (Sauer et al., 1995). In animals,
endoreduplication is only observed in specific cell types
such as some molluscan neurons, mammalian megakaryocytes and placental trophoblast cells (Chase and
Tolloczko, 1987; Zybina and Zybina, 1996). The function
of endoreduplication is unknown, although some of the
proposed roles include gene amplification, radiation
resistance and cell differentiation (Nagl, 1976; Barlow,
1978; Galbraith et al., 1991).
The occurrence of endoreduplication is common in
plants (Barlow, 1978). Ploidy level of plant cells has
been determined by using conventional cytophotometric
methods since the early 1950s (D’Amato, 1984). Many
informative investigations have been made in specific cell
types that are highly specialized, such as suspensor cells in
Phaseolus (Nagl, 1978), basal cells of the hairs on Bryonia
anthers (Barlow, 1975) and storage parenchyma cells in
cotyledons of several legume species (Smith, 1971; Millerd
and Whitfield, 1973; Davies, 1976). The former two types
of cells represent examples of polytene chromosomes.
However, the cytophotometric analyses have been limited
to a relatively small number of nuclei and simply showed
the presence or absence of one or two genomic copies
of DNA (DeRocher et al., 1990). Flow cytometry has
opened the way for rapid and accurate determination of
C levels from a large number of nuclei (Galbraith, 1983).
Recently, flow cytometric data for the systemic control of
endoreduplication have been accumulated in economically important crops, such as cucumber (Gilissen et al.,
1993), tomato (Smulders et al., 1994) and Brassica crops
(Kudo and Kimura, 2001a). A more recent investigation
reported detailed patterns of endopolyploidy in various
vegetative tissues in cabbage plants (Kudo and Kimura,
2001b). In this case, the number of endoreduplication
To whom correspondence should be addressed. Fax: q81 270 20 8016. E-mail: [email protected]
ß Society for Experimental Biology 2002
1018
Kudo and Kimura
cycles (endocycles) is tissue-specific and is characteristic
of the stage of development.
Numerous observations have been made of endoreduplication, but the function of this process has not been
thoroughly investigated. Recently, there were studies
directed at the molecular mechanisms responsible for
the endoreduplication cycle in maize endosperm (Larkins
et al., 2001). Grafi and Larkins have shown that endoreduplication of maize endosperm involves the inhibition
of the M-phase-promoting factor and the induction of
S-phase-related protein kinase (Grafi and Larkins, 1995).
This implies that in plants as well as Drosophila, the
transformation of the endoreduplication cycle requires
down-regulation of the mitotic cycle. Grafi et al. also
identified a member of the retinoblastoma family,
which in animals controls cell proliferation and also
endoreduplication (Grafi et al., 1996).
In this study, the aim was to document endoreduplication events in the course of petal development of cabbage.
The temporal and spatial regulation of the mitotic activity
of epidermal tissues was investigated and nuclear DNA
synthesis was measured as determined by flow cytometry
during the different stages of petal development. It was
shown that differentiating cells in the proximal part of the
petal become more polyploid as the mitotic activity of
the petal epidermis decreases, unlike in the distal part of
the petal. The differentiated epidermal cells toward the
base of the petal are large and elongated, having extremely
large nuclei. A relationship was proposed between ploidy
levels and cell size during petal development.
Materials and methods
Plant materials
Cabbage (Brassica oleracea L. var. capitata cv. Ri-Nen) was
used. Plants were grown to maturity in a greenhouse under
natural conditions. When the plants flowered, buds of different
developmental stages were removed from the main inflorescence
and the petals were dissected from the flower bud under a
binocular microscope.
1 mg ml1 for 20 min. The epidermis was then washed in distilled water. Stained DNA was then visualized by epifluorescence microscopy (model BHS-RFC, Olympus) using the 3 20
and 3 40 objectives of a microscope. A total of 1000 nuclei
were counted for each developmental stage.
Nuclei isolation and flow cytometric analysis
Nuclei were prepared from petals harvested at different developmental stages with a high resolution kit (Partec High Resolution
Kit type P, Partec GmbH, Münster, Germany) according to the
manufacturer’s instructions. Whole petals or dissected tissues
were chopped with a sharp razor blade in 0.5 ml of nuclei
extraction buffer (solution A of the kit). After filtration through
a 30 mm Cell Trics filter (Partec), 0.5–2.0 ml of staining solution
containing the dye DAPI (solution B of the kit) was added. The
analyses were performed with a PAS flow cytometer (Partec)
equipped with an HBO lamp for UV excitation. Fluorescence
peaks were estimated with the WinMDI software (version 2.8,
copyright ß 93–99 Joseph Trotter). Cytometric analysis was
performed on a minimum of 103 nuclei (total count). Measurements of nuclear DNA content were carried out with at least six
replications originating from different buds and mature flowers.
To determine the standard peak position of 2C nuclei, the 2C
peak from nuclei of in vitro young leaves was analysed at least
twice on each measurement. The data were plotted on a semilogarithmic scale, so that the histogram peaks from 2C to 32C
were evenly distributed along the abscissa. The data were
presented as a percentage of the total amount of nuclei in all
peaks of the histogram.
Results
Development of cabbage petals
The developmental stages of cabbage petals are shown
in Fig. 1. Five stages were morphologically distinguished
from young petals (stage 1) to mature petals (stage 5). At
stage 1, petals have formed small, flat laminae, which
reached to about two-thirds of stamen height. At stage 5,
Cell size measurement
Petals were fixed in a solution of 3 parts 95% ethanol to 1 part
acetic acid for 4 h at room temperature and stored in 70%
ethanol at 4 8C. Fixed tissues were soaked in water and then
incubated overnight in chloral hydrate solution (25 g in 25 ml).
Adaxial epidermal cells were examined using a confocal
laser-scanning microscope (model Fluoview-BX50, Olympus).
Cell surface areas were determined using a Fluoview image
analysis system (Olympus).
DNA staining and mitotic index determination
Mitotic indexes were determined on the adaxial epidermis of
petals harvested at different stages of development. The epidermis was placed on a glass slide and immersed in a drop
of 49,6-diamidino-2-phenylindole (DAPI) at a concentration of
Fig. 1. Five stages in cabbage petal development used in this study.
Stage 1, petals dissected from young flower buds 4 mm in length, and
stage 5, mature petal. Bar ¼ 5 mm.
Endoreduplication in cabbage petal development
the structure of the fully expanded petals was relatively
simple and a vascular array was apparent in the petals
(Fig. 1). Analysis of epidermal cell area in relation to
position in the mature petal (Fig. 2) showed that in the
flattened distal part of the petal, the epidermal cells were
small and homogeneous. By contrast, the epidermal cells
toward the base of the petal were large and extremely
elongated. These two types of epidermal cells each
occupied about 50% of the petal length and the point of
transition of epidermal cell shape is quite marked (Fig. 2).
The elongate morphology of the proximal epidermal cells
appear to relate to their rapid expansion which pushes the
petal out of the bud as it opens. The distal epidermal cells
did not expand significantly during the latter stages of petal
development (stage 3 and stage 5), whereas the proximal
epidermal cells increased, on average, approximately
6-fold in cell surface area (Fig. 2). The mitotic index
in the petal epidermis sharply decreased during petal
development (Fig. 3). The mitotic index in the distal part
of the petal remained at stage 3. From these observations
of mitotic figures between stage 3 and full expansion
(stage 5), there was no evidence for cell division in the
proximal part of the petal (data not shown), and the
increase in petal size during this period appears to be
mainly as a result of cell expansion in the proximal part
of the petal.
Fig. 2. The relationship between the surface area of epidermal cells
and their distance from the petal base at different developmental stages.
(m), stage 3 and (k), stage 5.
1019
Fig. 3. The mitotic index in epidermal cells in cabbage petal development. (A) Whole petals between stage 1 and stage 2, and (B) the distal
part of the petal between stage 3 and stage 5.
Fig. 4. Fluorescence of DAPI-stained nuclei in epidermal cells of mature
cabbage petals. (A) Nuclei from the distal part of the petal. (B) Nuclei
from the proximal part of the petal. Bars ¼ 20 mm.
1020
Kudo and Kimura
The observation of DAPI-stained petal tissues revealed
apparent differences in nucleus size in relation to position
in the mature petal (Fig. 4). Epidermal cells in the
distal part of the petal contained small nuclei (Fig. 4A),
whereas elongated epidermal cells in the proximal part
of the petal had large nuclei (Fig. 4B).
Increase of ploidy level during petal development
Figure 5 shows the evolution of the ploidy level during
petal development. For earlier stages (stage 1 and stage
2), whole petals were used for nuclear preparations, as the
petals were too small to separate the different parts. From
stage 3 to stage 5, the petals were dissected into two parts,
the proximal part and the distal part. In very young
developing stages (from stage 1 to stage 2), petals
exclusively contained nuclei with the 2C and 4C DNA
levels, accounting for 46% (2C) and 54% (4C) of total
nuclei at stage 1, and 37% (2C) and 63% (4C) of total
nuclei at stage 2. Because cabbage is a diploid species
(2n ¼ 18), the 2C DNA level corresponds to the diploid
state of the genome found in the G1 phase, whereas the
4C DNA level results from the S-phase doubling of
chromatids found in the G2 phase, and thus is an
indicator of the capacity of cells to enter mitotic cycles.
Therefore, the major 2C and 4C cells suggest that the
tissue is in a dividing state. Later in development (from
stage 3 to stage 5), cells in the proximal parts of the petal
significantly increased their ploidy level, but cells in the
distal part of the petal maintained diploid level. The first
cycle of endoreduplication of nuclear DNA had taken
place at stage 3 as indicated by a small proportion
of polyploid 8C nuclei. Further endoreduplication had
occurred at stage 4. At this stage, the proximal part of the
petal tissue contained cells with 16C levels, but nuclei
at the 2C DNA level were still detectable. An additional
round of endoreduplication was detected at stage 5.
About 5% of cells of the basal part of the petal had
undergone four rounds of endoreduplication cycles,
reaching the 32C ploidy level. The flow cytometric profiles
displayed a small but reproducible 32C peak from nuclei
in the proximal part of the petal at stage 5 (Fig. 6B).
Fig. 5. The evolution of the ploidy level during cabbage petal development. (A) Early stages (stage 1 and stage 2) of petal development, (B) late stages
(stage 3, stage 4, and stage 5) of petal development, the distal part of petal, and (C) late stages of petal development, the proximal part of petal.
Endoreduplication in cabbage petal development
At this stage, in contrast, the number of nuclei at
the 2C DNA level decreased dramatically and became
undetectable (Fig. 6B). However, nuclei from the distal
part of the petal gave the 2C and 4C peaks from stage 3
to stage 5 (Figs 5, 6A).
Discussion
The study of cabbage petals may help in providing
information about the relationship between endoreduplication and cell growth during plant organ development.
In the present study, one of the most striking features of
petal cells is the uneven increase of their DNA content
during development. This cytometric data showed that,
in the proximal part of the petal, cells become more
endopolyploid with up to a 32C ploidy level, whereas cells
in the distal part of the petal maintained the diploid level
throughout petal differentiation. At stage 5, flow cytometric profiles for the distal part of the petal is characterized by a high proportion of nuclei at the 2C and 4C
Fig. 6. Characteristic histograms of nuclei distribution at stage 5.
(A) Nuclei from the distal part of the petal, (B) nuclei from the proximal
part of the petal.
1021
levels, indicating that the cells never endoreduplicate and
their mitotic cell cycle is arrested either in the G1 phase
or in the G2 phase of the diploid cell cycle, even at the
mature stage of the flower. The results suggest that
the occurrence of endoreduplication may be not only
temporally, but also spatially regulated in a whole petal.
At the mature stage, epidermal cells in the proximal
part of the petal are large and extremely elongated, but
epidermal cells in the distal part of the petal are small and
highly homogeneous. These findings, together with microscopic observations of nuclei, indicate that the formation of large differentiated cells is accompanied by an
increased ploidy level. The correlation between the cell
size and the degree of endopolyploidy supports the idea
that the nuclear DNA content might play a key role in
controlling cell size. Endoreduplication in cabbage petals
might be a major driving force for cell differentiation. By
measuring the mitotic index of epidermal cells of petals,
it was shown that the distribution of mitotic activity
is determined not only temporally, but also spatially
within a whole petal and the results suggested that DNA
synthesis continues in the basal petal cells after cell
division has ceased.
The correlation between DNA contents and cell size
is not known: however, on the basis of these results, a
possible scenario explaining the correlation between
ploidy levels and cell size during petal development is
presented in Fig. 7. Petal tissues can be considered as
being composed of cells that simultaneously react upon
developmental cues. During petal development, at
least two consecutive developmental signals provoke two
simultaneous responses of the cells; the first one triggering
the arrest of the cell cycle, and the second one triggering
the onset of endoreduplication. These signals may be
related to endogenous positional cues causing the distinct zonation. In the distal part of the petal, cells never
endoreduplicate. Upon receiving the arrest signal of the
cell cycle, the cells will increase in size until cell expansion
Fig. 7. Scenario explaining the correlation between ploidy levels and
cell size during development of cabbage petal. (A) In the distal part
of the petal, cells never endoreduplicate, and (B) in the proximal part
of the petal, cells can switch to endoreduplication.
1022
Kudo and Kimura
is arrested simultaneously. This results in a homogeneous
final cell size distribution, as observed in the distal part of
the petal (Fig. 7A). In the proximal part of the petal, cells
can switch to endoreduplication. The switch to endoreduplication cycles is not necessarily synchronous for
neighbouring cells. As a result, size differences will have
built up by the time the cells receive the arrest signal of
the cell cycle. These differences will be amplified during
the petal expansion phase, and as a result, the final cell
size will be correlated with ploidy levels (Fig. 7B).
Extensive endoreduplication has recently been detected
in cabbage flowers (Kudo and Kimura, 2001c). The
investigators showed the presence of endopolyploidy in
different organs of cabbage flowers. For instance, filament tissues contain cells with six ploidy levels (2C–64C)
and carpel tissues have cells with three ploidy levels
(2C–8C). The 2C–32C range of nuclear ploidy in cells
of cabbage petal has been also described. These findings,
together with the data presented in this study, indicate
that endoreduplication is a common feature in the development of cabbage flowers. However, endoreduplication is
not obvious in Arabidopsis thaliana, a related crucifer to
Brassica species. A. thaliana lacks somatic polyploidy in
floral organs in which all cells are at the 2C level
(Galbraith et al., 1991).
A coupling between cell size and nuclear DNA content
has been documented in many eukaryotic organisms.
Cells that have undergone endoreduplication are larger
than comparable cells that have not (Larkins et al., 2001).
In budding yeast, Saccharomyces cerevisiae, diploid cells
are larger than haploid cells (Sherman, 1991). In fission
yeast, Schizosaccharomyces pombe, mutant cells that have
undergone successive endoreduplication rounds become
gigantic (Moreno and Nurse, 1994). In plants, there is a
body of evidence that the size of cells is linked to their
DNA content (Kondorosi et al., 2000). This is illustrated
by the correlation between cell surface and nuclear DNA
levels in neighbouring leaf epidermal cells of Arabidopsis
(Melaragno et al., 1993) and maize (Cavallini et al., 1997).
Arabidopsis mutants that increase or reduce the ploidy
levels in trichomes invariably show an increase or a
reduction of the final cell size (Folkers et al., 1997). These
results also support the idea that endoreduplicaton
is coupled with cell size. Endoreduplication levels could
determine cell size in that endoreduplication occurs
before the onset of the massive expansion of petals
in flower opening. However, endoreduplication is not
always coupled with an increase in cell size and can be,
at least partially, uncoupled from cell elongation: when
Arabidopsis seedlings are allowed to germinate, endoreduplication takes place before the elongation of
hypocotyl cells (Gendreau et al., 1997).
The correlation between ploidy levels and cell size
indicates that endopolyploid nuclei might be required
for the formation of large cells. The physiological role
of developmentally programmed polyploidy is, however,
elusive. Multiplication of the genome has been proposed
to increase metabolic activity, rRNA synthesis and
transcriptional activity (Baluska and Kubica, 1992).
Endopolyploid nuclei can be advantageous for specialized
functions. During maize endosperm development, cells
shift from mitotic cycles to endoreduplication cycles,
driving the massive synthesis of storage proteins (zein)
and starch (Lur and Setter, 1993). The extent of endopolyploidy correlates with the yield of maize grain (Kowles
et al., 1992; Cavallini et al., 1995). Endoreduplication
in the pea cotyledons is related to the accumulation of
storage products (Liu et al., 1996).
Endopolyploidy may be important for understanding
the regulation of gene expression in differentiated tissues.
Galitski et al. described the impact for polyploidy on gene
expression in isogenic budding yeast S. cerevisiae strains
that varied in ploidy from haploid to tetraploid (Galitski
et al., 1999). The authors showed that a subset of genes
was induced or repressed in a ploidy-dependent manner.
Recent work on Medicago root nodules provided molecular evidence for the regulation of cell size by endopolyploidy (Cebolla et al., 1999). The work demonstrated
a gene ccs52, a plant homologue of the anaphasepromoting complex activators that are involved in
mitotic-cyclin degradation. Overexpression of this gene
in fission yeast resulted in the arrest of mitosis, and
induced endoreduplication and cell enlargement.
The developmental signals that control the exit from
the cell cycle or transform the mitotic cycle to endocycle
during cell differentiation have not yet been identified
(Kondorosi et al., 2000). However, plant cells can elect
different options for cell growth during differentiation
(Melaragno et al., 1993; Valente et al., 1998). Upon differentiation signals, plant cells can exit from the mitotic
cycle both in the G1 and G2 phases with 2C and 4C DNA
content, respectively. One option is to differentiate with
original 2C and 4C ploidy level (G1 and G2 differentiation). The growth potential and metabolic activity of 4C
cells during G2 differentiation is greater than those of
2C cells during G1 differentiation. The other option is to
enter the endocycle, resulting in the same nucleus with
increased ploidy levels. Duplication of the genome can
augment further the growth capacity and the extent of cell
enlargement. The control of endoreduplication may allow
cells to reach extraordinary sizes (Cebolla et al., 1999).
This process probably provides a means to manipulate
the cell size in agronomically important crops.
References
Baluška F, Kubica Š. 1992. Relationship between the content
of the basic nuclear proteins, chromatin structure, rDNA
transcription and cell size in different tissues of the maize
root apex. Journal of Experimental Botany 252, 991–996.
Endoreduplication in cabbage petal development
Barlow PW. 1975. The polytene nucleus of the giant cell of
Bryonia anthers. Protoplasma 83, 339–349.
Barlow PW. 1978. Endopolyploidy: towards an understanding
of its biological significance. Acta Biotheoretica 27, 1–18.
Cavallini A, Natali L, Balconi C, Rizzi E, Motto M, Gigot C,
D’Amato F. 1995. Chromosome endoreduplication in endosperm cells of two maize genotypes and their progenies.
Protoplasma 189, 156–162.
Cavallini A, Natali L, Cionini G, Balconi C, D’Amato F. 1997.
Inheritance of nuclear DNA content in leaf epidermal cells of
Zea mays L. Theoretical and Applied Genetics 94, 782–787.
Cebolla A, Vinardell JM, Kiss E, Olah B, Roudier F,
Kondorosi A, Kondorosi E. 1999. The mitotic inhibitor ccs52
is required for endoreduplication and ploidy-dependent cell
enlargement in plants. The EMBO Journal 18, 4476–4484.
Chase R, Tolloczko B. 1987. Evidence for differential DNA
endoreduplication during the development of a molluscan
brain. Journal of Neurobiology 18, 395–406.
D’Amato F. 1984. Role of polyploidy in reproductive organs
and tissues. In: Johri BM, ed. Embryology of Angiosperms.
New York: Springer-Verlag, 523–566.
Davies DR. 1976. DNA and RNA contents in relation to cell
and seed weight in Pisum sativum. Plant Science Letters 7,
17–25.
DeRocher EJ, Harkins KR, Galbraith DW, Bohnert HJ. 1990.
Developmentally regulated systemic endopolyploidy in succulents with small genomes. Science 250, 99–101.
Folkers U, Berger J, Hülskamp M. 1997. Cell morphogenesis
of trichomes in Arabidopsis: differential control of primary
and secondary branching by branch initiation regulators and
cell growth. Development 124, 3779–3786.
Galbraith DW. 1983. Rapid flow cytometric analysis of the cell
cycle in intact plant tissues. Science 220, 1049–1051.
Galbraith DW, Harkins KR, Knapp S. 1991. Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiology 96,
985–989.
Galitski T, Saldanha AJ, Styles CA, Lander ES, Fink GR. 1999.
Ploidy regulation of gene expression. Science 285, 251–254.
Gendreau E, Traas J, Desnos T, Grandjean O, Caboche M,
Höfte H. 1997. Cellular basis of hypocotyl growth in
Arabidopsis thaliana. Plant Physiology 114, 295–305.
Gilissen LJW, van Staveren MJ, Creemers-Molenaar J,
Verhoeven HA. 1993. Development of polysomaty in seedlings
and plants of Cucumis sativus L. Plant Science 91, 171–179.
Grafi G, Burnett RJ, Helentjaris T, Larkins BA, DeCaprio JA,
Sellers WR, Kaelin Jr WG. 1996. A maize cDNA encoding
a member of the retinoblastoma protein family: involvement
in endoreduplication of S phase-related kinases. Proceedings
of the National Academy of Sciences, USA 93, 8962–8967.
Grafi G, Larkins BA. 1995. Endoreduplication in maize
endosperm: involvement of M phase-promoting factor
inhibition and induction of S phase-related kinases. Science
269, 1262–1264.
Kondorosi E, Roudier F, Gendreau E. 2000. Plant cell-size
control: growing by ploidy? Current Opinion in Plant Biology
3, 488–492.
1023
Kudo N, Kimura Y. 2001a. Flow cytometric evidence for
endopolyploidy in seedlings of some Brassica species.
Theoretical and Applied Genetics 102, 104–110.
Kudo N, Kimura Y. 2001b. Patterns of endopolyploidy during
seedling development in cabbage (Brassica oleracea L.).
Annals of Botany 87, 275–281.
Kudo N, Kimura Y. 2001c. Flow cytometric evidence for endopolyploidization in cabbage (Brassica oleracea L.) flowers.
Sexual Plant Reproduction 13, 279–283.
Kowles RV, McMullen MD, Yerk G, Phillips RL, Kraemer S,
Srienc F. 1992. Endosperm mitotic activity and endoreduplication in maize affected by defective kernel mutations.
Genome 35, 68–77.
Larkins BA, Dikes BP, Dante RA, Coelho CM, Woo Y-M, Liu Y.
2001. Investigating the hows and whys of DNA endoreduplication. Journal of Experimental Botany 52, 183–192.
Liu CM, Johnson S, Hedley CL, Wang TL. 1996. The generation
of a legume embryo: morphological and cellular defects in pea
mutants. In: Wang TL, Cuming A, eds. Embryogenesis: the
generation of a plant. Oxford: Bios, 191–213.
Lur H-S, Setter TL. 1993. Role of auxin in maize endosperm
development. Plant Physiology 103, 273–280.
Melaragno JE, Mehrotra B, Coleman AW. 1993. Relationship
between endopolyploidy and cell size in epidermal tissues of
Arabidopsis. The Plant Cell 5, 1661–1668.
Millerd A, Whitfield PR. 1973. Deoxyribonucleic acid and
ribonucleic acid synthesis during the cell expansion phase
of cotyledon development in Vicia faba L. Plant Physiology
51, 1005–1010.
Moreno S, Nurse P. 1994. Regulation of progression through the
G1 phase of the cell cycle by the rum1q gene. Nature 367,
236–242.
Nagl W. 1976. DNA endoreduplication and polyteny understood as evolutionary strategies. Nature 261, 614–615.
Nagl W. 1978. Endopolyploidy and polyteny in differentiation
and evolution. Amsterdam: Elsevier.
Pearson MJ. 1974. Polyteny and the functional significance of
the polytene cell cycle. Journal of Cell Science 15, 457–459.
Sauer K, Knoblich JA, Richardson H, Lehner CF. 1995. Distinct
modes of cyclin Eucdc2c kinase regulation and S-phase control in mitotic and endoreduplication cycles of Drosophila
embryogenesis. Genes and Development 9, 1327–1339.
Sherman F. 1991. Getting started with yeast. Methods in
Enzymology 194, 3–21.
Smith DL. 1971. Nuclear changes in the cotyledons of Pisum
arvense L. during germination. Annals of Botany 35, 511–521.
Smulders MJM, Rus-Kortekaas W, Gilissen LJM. 1994.
Development of polysomaty during differentiation in diploid
and tetraploid tomato (Lycopersicon esculentum) plants. Plant
Science 97, 53–60.
Valente P, Tao W, Verbelen J-P. 1998. Auxins and cytokinins
control DNA endoreduplication and deduplication in single
cells of tobacco. Plant Science 134, 207–215.
Zybina EV, Zybina TG. 1996. Polytene chromosomes in
mammalian cells. International Review of Cytology 165,
53–119.