Biotech Center for Agriculture and The Environment, Rutgers The State University of New Jersey, New Brunswick, NJ 08901, USA
* Author for correspondence (e-mail: lam{at}aesop.rutgers.edu)
Accepted 21 February 2003
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Summary |
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Key words: Endoreduplication, Polyploidy, Chromatin dynamics, GFP, Arabidopsis
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Introduction |
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We previously demonstrated the feasibility of detecting and tracking
specific insertion sites in live Arabidopsis plants with the LacI-GFP
+ lac-operator tagging system
(Kato and Lam, 2001). In this
approach, concatameric arrays of lac operators (256-mer, 10.1 kbp)
were randomly inserted into chromosomes along with a glucocorticoid-inducible
system that drives expression of a fusion protein that consists of GFP, LacI
DNA-binding domain and the SV40 T-antigen nuclear localization signal
(GFP-LacI/NLS; Fig. 1a). Upon
induction of GFP-LacI/NLS protein, we can detect the tagged loci as
GFP-binding fluorescent spots. We showed that addition of
isopropyl-ß-D-1-thiogalactopyranoside (IPTG), which inhibits LacI binding
to the lac operator, significantly decreased the number of detectable
GFP spots. We also showed that the induced GFP-LacI/NLS fusion proteins do not
localize to specific sites when the lac operator array is absent from
the genome. These controls demonstrated that there are few artefacts in our
system, in which 1-2-week-old seedlings with a size range of 5-10 mm are
directly mounted on microscope slides with water as the mounting medium
(Fig. 2a). Under these
conditions, Arabidopsis seedlings can remain viable for at least 24
hours and can grow normally upon their return to soil.
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In this work, we measured chromatin dynamics in interphase plant cells.
This is the first report of such studies for live plants, to our knowledge.
Moreover, we tested whether chromatin dynamics change in different cell types.
In Drosophila, the confinement area of spermatocyte nuclei
(Vazquez et al., 2001) is much
larger than that of stage 12 embryos
(Marshall et al., 1997b
).
However, it is not known whether these differences are a reflection of cell
type or the GFP-tagged loci examined. In this work, we compared chromatin
dynamics of specific loci in different cell types of the same transgenic
plants so that we can eliminate locus- and sample-related variations. We chose
to compare guard cells and pavement cells for two reasons. First, the two cell
types are both located on the epidermis of the seedlings and can be
distinguished by their shapes (Fig.
2b). Therefore, we could observe the nuclei of these cells with
minimal optical perturbation. Second, most, if not all, the guard cells are
diploid whereas most pavement cells are polyploid owing to DNA
endoreduplication (Melaragno et al.,
1993
). Therefore, we might be able to correlate the effects of
endoreduplication with changes in chromatin dynamics.
Endoreduplication is a phenomenon in which chromosomes duplicate without
any obvious condensation and decondensation steps, leading to the production
of endopolyploid cells with enlarged nuclei. Endoreduplication is widely
observed in the animal and plant kingdoms, although it is more common among
insects and angiosperms that have small genomes
(Nagl, 1976). Constant
tissue-specific patterns of endoreduplication in different organs and cell
types suggest that endoreduplication is an essential part of the developmental
programs in post-mitotic cells (Mizukami,
2001
). In fact, ploidy levels affect gene expressions in budding
yeast (Galitski et al., 1999
)
and plants (Lee and Chen,
2001
). It was of interest to us to study the effects of
endoreduplication on chromatin dynamics because it could provide clues to the
mechanisms that mediate changes in gene expression in polyploid cells
(Edgar and Orr-Weaver, 2001
;
Madlung et al., 2002
).
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Materials and Methods |
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Genomic library screening
Genomic DNA of homozygous EL702C Arabidopsis plant was extracted
and digested with BglII and HindIII to release the T-DNA
left border region from the lac repressor expression cassette
together with genomic DNA flanking the insertion sites. DNA fragments of 2-5
kbp were used to construct the partial genomic DNA library with a ZAP Express,
BamHI-predigested Gigapack cloning kit (Stratagene Cloning Systems,
CA). The library (2x105 pfu) was screened with radiolabeled
T-DNA left border region DNA fragments
(Kato and Lam, 2001).
Adaptor PCR screening
BglII and HindIII-digested genomic DNA fragments of 2-5
kbp were eluted from an agarose gel and ligated to a BamHI cassette
that is provided with the LA-PCR in vitro cloning kit (Takara, Japan). PCR was
performed according to the manufacturer's protocol. Oligonucleotides
(5'-ATGTCTCTGACCAGACACCCATCAACAGTA-3' and
5'-GGGATCCTGTACTCCACCAAAGAAGAAGAG-3') whose sequences are present
in the T-DNA left border region were used as defined region-specific
primers.
Sample preparation for microscopy
Surface-sterilized seeds were germinated on 0.5x MS agar plates.
1-2-week-old seedlings were transferred to fresh 0.5x MS agar plates and
40 µl of a 0.3 µM dexamethasone (Dex) solution was then dropped on each
seedling. After 10-12 hours, the seedlings were placed between two coverslips
under water. The coverslips were then placed on the microscope stage for
microscopic imaging.
DAPI staining
We followed the method described by Melaragno et al.
(Melaragno et al., 1993).
Homozygous EL702C seedlings were fixed in a solution of three parts 95%
ethanol to one part glacial acetic acid for 2 hours at room temperature and
then transferred to 70% ethanol. Fixed cells were then soaked in water and
transferred into 0.5 M EDTA containing 5 µg ml1 of
4',6'-diamidino-2-phenylindole (DAPI) for 1 hour. The stained
seedlings were washed with water and set on the microscope slides as described
above. Nuclear images were obtained with a 60x water-immersion objective
lens and two-dimensional non-deconvolved nuclear images were used to obtain
the fluorescence intensities. The mean intensity of three nuclei in root tip
cells was used to compare the relative intensities of guard cell and pavement
cell nuclei. Three independent samples were used.
Fluorescence microscopy and spot detection
A DeltaVision restoration microscope system (Applied Precision, WA)
equipped with a TE200 microscope (Nikon, Japam) was used to observe nuclei in
Dex-induced seedlings. 40 images at 0.2 µm z-axis steps were
collected using a Nikon PlanApo 60x 1.2 NA water-immersion objective
lens. The exposure times were 0.3-3.0 seconds and the filters used were:
exciter, 436 nm/10 nm; emitter, 470 nm/30 nm; and a JP4 beamsplitter (Chroma,
VT). The images presented were deconvolved based on a point-spread function
data.
Because a relatively high accumulation of GFP is required to visualize
spots in the nucleus, the background-to-signal (free versus bound GFP fusion
protein) ratio is a significant factor. High levels of GFP accumulation might
also cause nonspecific GFP aggregation. Thus, the nuclei were observed under
conditions with minimal GFP accumulation (6-12 hours after induction with 0.3
µM Dex) and lac-operator-binding GFP spots were defined
statistically. In this work, the spots were defined as a cluster of pixels
that contained outliers in images of the observed nuclei. The intensity values
(16-bit integer) of each pixel in optically z-axis-sectioned images
were measured and the outlier is defined as:
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Measurement of nuclear volumes
Volumes of the nuclei were estimated by measuring diffused GFP background
fluorescence in the nuclei, which were used to measure chromatin movements, at
the first time point. Deconvolved three-dimensional (3D) nuclear images were
analysed to obtain the volumes. GFP signals were selected in each optical
section and the volumes were calculated as a sum of GFP signal areas in a
total of 40 sections.
Measurement of chromatin movement
Overall mean-square differences in distance between GFP spots
<d2> plotted against elapsed time intervals
t in guard-cell nuclei of cotyledons from EL702C homozygous
plants are measured. When the data on the 12th (11 minutes) and 13th (12
minutes) time points were collected, the exciter shutter was closed from the
3rd to 10th points in order to minimize photobleaching of the samples.
Plants were fixed by treatment with ice-cold methanol for 5 minutes followed by ice-cold acetone for 30 seconds. The seedlings were immediately dried with paper towels and floated in a Petri dish with distilled water until it was set on the microscope slide.
Image processing
The stacked images of nuclei were analysed by softWoRx software (Applied
Precision, WA) on an Octane Workstation (Silicon Graphics, CA). The images
were then processed by Adobe Photoshop 5.5 (Adobe Systems, CA) on a PowerMac
G4 computer (Apple, CA) for the final images.
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Results |
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Comparative analysis of nuclear properties for guard cells and
pavement cells in homozygous EL702C seedlings
In Arabidopsis thaliana, ploidy levels for cells can vary from 2C
(stomatal guard cells, sepal and petal epidermal cells) to 64C (leaf trichome)
(Melaragno et al., 1993).
Guard cells are components of stomata that regulate gas exchange and are
distributed across the leaf and hypocotyl epidermis with characteristic
spacing. These cells develop in pairs symmetrically from common mother cells
and their cell cycle is arrested in G1 phase after differentiation
(Melaragno et al., 1993
). The
shape of the differentiated cell is a uniformed crescent shape
(Fig. 2b), whereas their
nucleus is usually disk-like (Fig.
2d). In contrast to guard cells, pavement cells fill the surface
of hypocotyl epidermis and grow in one direction without cell division,
resulting in elongated, rectangular shapes
(Fig. 2c). These cells contain
a large nucleus that is usually elongated along the axis of the hypocotyl, and
mature pavement cells have typically undergone several rounds of
endoreduplication (Fig.
2e).
We first characterized the nuclei of guard cells and pavement cells in
homozygous EL702C seedlings by fluorescence microscopy after Dex induction. In
guard-cell nuclei, the spot numbers observed upon Dex treatment vary from none
(not detected) to four (Fig.
2d, Fig. 3a);
40% of the guard cells in homozygous lines showed two spots. In pavement
cell nuclei, the spot numbers were more variable and
10% of cells showed
up to eight spots (Fig. 2e,
Fig. 3a).
|
To verify the expected ploidy difference between guard cells and pavement
cells, the relative quantities of DNA in nuclei of these two cell types were
measured by DNA-specific DAPI staining. Fluorescent intensities of each
nucleus were compared with the average intensities of root-tip-cell nuclei, in
which the ploidy level is known to be 2C
(Melaragno et al., 1993).
Fluorescent intensities of guard-cell nuclei in the homozygous EL702C line are
the same as root-tip-cell nuclei (Fig.
3b). By contrast, DAPI fluorescence intensities in pavement cell
nuclei are two to eight times higher than root-tip cells, and the average
intensity is approximately five times higher than that of guard cells
(Fig. 3b). These results are
consistent with previous studies
(Melaragno et al., 1993
). The
difference in nuclear volumes between guard cells and pavement cells was
determined by calculating the volume of diffuse GFP background fluorescence
caused by unbound fusion proteins in 3D models of the nuclei
(Kato and Lam, 2001
). In guard
cells, nuclear volumes are
50 µm3, whereas the volumes of
nuclei in pavement cells are
250 µm3 on average
(Fig. 3c). The positive
relationship between nuclear volume and ploidy levels is consistent with
previous findings (Baluska,
1990
) and shows that the guard cells and the pavement cells we
observed display the expected degree of ploidy. In this study, we did not
measure the GFP spot numbers in the same samples that were used for DAPI
staining because DAPI staining affected GFP-LacI/NLS induction and might also
affect the viability of the samples. Therefore, we could not directly address
the relationship between GFP spot numbers and ploidy level within the same
nuclei.
Chromatin dynamics in guard cells and pavement cells
To compare chromatin dynamics in diploid and endoreduplicated nuclei, we
characterized the motion of the inserted loci in nuclei of guard cells and
pavement cells in live EL702C Arabidopsis seedlings after Dex
induction. Chromatin motions were represented by plotting the overall mean
squared change in distance <d2> between two
spots against elapsed time interval
t
(Marshall et al., 1997b
). For
two freely diffusing objects undergoing random-walk motion, a plot of
<
d2> against
t should
increase continuously if the movement is not constrained. If the movement is
constrained to a certain area, <
d2> should
become independent of
t over time and the plot will
plateau.
First, we measured the chromatin movement in guard cells. We detected two
spots in most of the analysed nuclei. We measured the distance between pairs
of spots per nucleus as a function of time. As a total, we measured nine pairs
of spots in a total of nine nuclei. The plot of measurements taken from live
plants produced <d2> values that essentially
remain unchanged in the range of 10 minutes
(Fig. 4). We compared the plot
to that observed with chemically fixed plants and found that the value of
<
d2> for live cells showed an approximately
three times higher range at all time points measured, demonstrating that the
movements are not due to experimental error or instrument noise. The
asymptotic <
d2> value of 0.03
µm2 (Fig. 4) indicates a mean change in distance between two spots of 0.17 µm. The
maximum radius of the constrained area for guard cell nuclei, which was
obtained by measuring
d for various time points from
independent experiments (data not shown) was 0.21 µm. The results indicate
that the tagged loci in the transgenic line undergo diffusive movement in
constrained areas within the nucleus. For particles undergoing 3D random walks
with a diffusion constant D, it can be shown that a plot of
<
d2> against
t should
increase linearly with a slope of 4D
(Marshall et al., 1997b
).
Thus, one can estimate the diffusion constant based on the plots. In
Drosophila spermatocytes, diffusion coefficients of early G2 phase
chromatin are 1.0x103 µm2
s1 and 0.94x104 µm2
s1 in late G2 phase. At the present time, with our
experimental system, it takes
50 seconds to detect the tagged loci with
high signal-to-noise ratios using 3D restoration microscopy. Thus, we are
unable to perform more rapid time-lapse measurements of the relative distance
between two tagged loci. We consequently could not measure precisely the
earlier phase in the plot of <
d2> versus
t in order accurately to determine the precise diffusion
coefficient for individual tagged loci. However, because the asymptotic value
of <
d2> (0.03 µm2) was
apparently reached in less than 1 minute, we estimate this would put a lower
limit of 1.25x104 µm2
s1 for D, assuming that the plot of
<
d2> against
t reaches a
plateau at 1 minute in a linear fashion.
|
Next, we measured the chromatin movement of pavement cell nuclei by a plot
of the overall mean squared change in distance
<d2> between two spots for the elapsed time
interval
t (Fig.
4). The spot numbers varied between three and six spots in
analysed nuclei. In most cases, `spot numbers 1' pairs were measured
for each of the nuclei studied. As a total, we measured 36 pairs of distances
in ten nuclei. We also performed similar measurements with pavement cell
nuclei that were chemically fixed to confirm that the different movements from
guard cell nuclei are not caused by altered conditions when dealing with
different cell types in different parts of the plant. Plots of measurements
made with pavement cell nuclei show that <
d2>
increases until 7 minutes (
t) and reached a plateau of 0.10
µm2, which indicates a mean change in distance between the spots
of 0.32 µm. The maximum radius of the confinement area, which was also
obtained by measuring
d for various time points from
independent experiments (data not shown), was 0.44 µm. Diffusion
coefficients calculated from the slope is 5.35x105
µm2 s1. We did not detect any correlation for
distances between the pairs of spots and the changes (i.e. there is no
evidence that spots close to each other move less than spots that are further
apart) in this work.
The Student's t-test for <d2>
values using a two-tailed distribution between the samples of diploid guard
cells and endoreduplicated pavement cells at 9 minutes and 10 minutes gave
P values of 0.0050 and 0.0054, respectively. This suggested that the
values of <
d2> at the plateau are
significantly different. Our results indicate that chromatin movement of
endoreduplicated pavement cell nuclei might have a lower diffusion coefficient
(<2.3 times) but larger constrained areas (6.6 times larger on average and
9.2 times as a maximum) than guard cell nuclei.
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Discussion |
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However, we can estimate the coherence probabilities of multiple chromatids
in endoreduplicated cells by comparing the average GFP spot numbers and the
average DAPI fluorescence intensities in endoreduplicated pavement cells
versus diploid guard cells. If all chromatids are separated, the ratio of GFP
spot numbers in pavement cells to guard cells would be the same as that for
DAPI fluorescence intensities. By contrast, if all chromatids remain cohered,
the GFP spot numbers would not increase (guard-cell to pavement-cell ratio of
1). Because the increment of GFP spot number ratio (2.5 times on average) is
half of that for the increase in DAPI fluorescence intensities (five times on
average) in this study (Fig.
3a,b), we predict that half of the chromatids might remain cohered
while the other half are separated in the endoreduplicated pavement cells.
This is consistent with the recent fluorescence in situ hybridization (FISH)
observation using fixed 4C nuclei with single-locus probes for chromosome 4.
The observation suggests that 50% of the chromatids in endoreduplicated
nuclei are not cohered (M. Klatte, V. Schubert and I. Schubert, personal
communication).
Chromatin attachment in plant interphase nuclei
In both guard cells and pavement cells, chromatin shows constrained
diffusion movement (Fig. 4).
This suggests that plant chromatin is tethered by static interactions within
nuclei, as found in other organisms (Chubb
et al., 2002; Heun et al.,
2001
; Ishii et al.,
2002
). The result allows studies of chromatin dynamics, which have
been done in yeast and animals, to be extended to plants with regards to the
mechanism of interchromosomal interactions. Lamin proteins are thought to
provide scaffolding structures that mediate the attachment of chromatin to the
nuclear envelope in animal cells (Gant et
al., 1999
; Lopez-Soler et al.,
2001
). However, lamins have not been identified in plants or the
budding yeast Saccharomyces cerevisiae
(Meier, 2001
). This fact and
our present finding suggest that plants and yeast might have a distinct set of
nuclear anchoring components that functionally replace lamins of animal
cells.
Factors that increase chromatin constrain areas in endoreduplicated
pavement cells
One of the features of endoreduplicated nuclei is increase in nuclear
volume (Baluska, 1990).
Although the biological consequences of the enlarged nucleus on nuclear
processes such as transcription are not clear
(Edgar and Orr-Weaver, 2001
), a
simple explanation of this feature is that nuclear volume is proportional to
ploidy levels to maintain a similar 3D space per haploid genome. The measured
increase in nuclear volume in pavement cells is approximately the same as the
estimated increase in DNA content relative to diploid guard cells
(Fig. 3b,c). Moreover, half of
the chromatids in endoreduplicated pavement cells are thought to be separated,
as discussed above. These features suggest that pavement-cell nuclei indeed
contain a similar (twofold difference in maximum) 3D space per haploid genome
as guard-cell nuclei.
By contrast, we found that the apparent confinement areas for chromatin
movement of endoreduplicated pavement-cell nuclei are over six times larger
than those found in guard-cell nuclei. This finding suggests that the
chromatin in endoreduplicated pavement cells has larger free spaces of
diffusion than that in guard-cell nuclei even though the chromosome space per
haploid genome is similar. What makes the confinement areas larger in pavement
nuclei than in guard cells? One simple explanation could be a reduction in the
concentration of proteins involved in chromatin organization in the larger
pavement-cell nuclei. This might result in an enlarged free space for
chromosome motion in these endoreduplicated cells, because of a decrease in
the number of anchoring sites or interactions for chromatin within the
nucleus. In fact, daughter nuclei of early G1 phase yeast cells, which are 40%
smaller than the mother-cell nuclei, show five-times-less-frequent large rapid
chromatin movements than mother cells in the same culture
(Heun et al., 2001).
Another observation that lends support to this explanation is the fact that
endoreduplicated cells of maize root show a decrease in staining with Fast
Green FCF, which stains basic proteins within the nuclei
(Balusuka and Kubica, 1992). An
alternative explanation is that the compositions of the chromatin-organizing
proteins are different between the two types of nuclei. Although the
functional significance of matrix-attachment motifs in both cis- and
trans-acting elements remains unclear, the importance of structural
features of cis-acting elements such as a narrow minor groove in
A/T-rich DNA has been suggested (Kas et
al., 1989
). It can be hypothesized that endoreduplicated nuclei
have a different type or sets of chromatin-organizing proteins from diploid
nuclei. Their different sequence affinity for anchoring chromatin and changes
in their quantities or composition could result in alteration of chromatin
dynamics. Therefore, it would be interesting to determine whether diploid and
endoreduplicated nuclei have different biochemical compositions in matrix
proteins.
Biological significance of increased confinement areas in pavement
cells
Changes of gene expression associated with ploidy differences in budding
yeast is not caused by alterations in growth rate or viability but instead by
an increase of DNA content (Galitski et
al., 1999). Allotetraploid (hybrid genome)-dependent changes in
gene expression of Arabidopsis have also been reported
(Lee and Chen, 2001
). In the
study, genes silenced in Arabidopsis suecica, an allotetraploid
derived from Arabidopsis thaliana and Cardaminopsis arenosa,
were investigated with an amplified fragment-length polymorphism (AFLP) cDNA
display method. The result indicates epigenetic regulation of orthologous
genes in polyploid genomes.
Epigenetic mechanisms can involve interchromosomal interactions. For
example, paramutation can silence allelic genes in heterozygous conditions
(Sidorenko and Peterson, 2001)
whereas transvection can change gene expression by combining an enhancer on
one chromosome and a promoter on another chromosome
(Wu and Morris, 1999
).
Marshall explained the relationship between interchomosmal interactions and
chromatin diffusion confinement as follows
(Marshall, 2002
). If two loci
are to interact physically, they must be located within the same 3D space in
the nucleus. However, because of non-random nuclear organization, not every
pair of loci will be equally likely to interact. Owing to constrained
diffusion, only those loci whose regions of confinement overlap are capable of
interacting; pairs of loci whose confinement regions do not overlap are unable
to interact because they cannot diffuse into contact.
Based on this functional model, we propose a model of developmental control
of gene expression in endoreduplicated cells: although the volume of
endoreduplicated nucleus is larger than that of diploid nucleus, the nuclear
space per haploid genome or chromosome territories stays similar. Therefore,
locus densities per nuclear space would be similar to those in diploid nuclei.
Endoreduplicated nuclei allow additional or reduced intra- and interchromatin
interactions by providing larger free spaces for chromatin diffusion. The
expansion in the effective confinement area in the same 3D space per haploid
genome (compared with the diploid nucleus) might arise from a decrease in the
concentration or a change in the properties of nuclear structural proteins. In
this manner, endoreduplicated cells might achieve a different gene expression
pattern from diploid cells through an epigenetic mechanism. This model might
also apply to the evolutionary strategy for species that carry small genomes
to modulate gene expression patterns in specific cell types, which has been
achieved in other species by varying DNA contents
(Nagl, 1976).
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Acknowledgments |
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