Characterisation of Arabidopsis transgenic lines with a
Dex-inducible transactivating overexpression system for
the study of cell division pattern in leaf morphogenesis
Hoe Han GOH
Candidate number: 050138401
Word count: 4495
050138401
Abstract
Debate on the role of cell division in leaf morphogenesis has been controversial but with the advent
of molecular technology which utilises the knowledge gained in cell-cycle regulation, it is now
possible to dissect the role of cell division with a temporal and spatial level of control. A YFP
fusion construct of a truncated cyclin-dependent kinase inhibitor, ICK1/KRP1109, which has
previously been shown to block cell entry into mitosis, was used in a pOpOn2 dexamethasoneinducible transactivating overexpression system to allow flexible inhibition of cell division. The
utility of the system depends upon the generation of a reliable transgenic line which stably express
the fusion construct. Two strategies have been taken in an attempt to optimise the use of the
pOpOn2 system: identification of a homozygous transgenic line with respect to resistance marker;
and by identifying the optimal concentration of dexamethasone (Dex) required for induction.
Segregation analysis and the quantification of YFP expression helped identify one transgenic line as
being homozygous for the kanamycin-resistance marker but unlikely for the fusion construct, and
counter-intuitively this line had a significantly lower expression of fusion construct than
heterozygous lines suggesting silencing. Eight seedlings across all four transgenic lines with the
highest level of expression according to the YFP analysis were chosen to be propagated. Different
concentrations of Dex, ranging from 0.01 to 10 μM, were tested through macroinduction
experiments on two transgenic lines. Concentration-dependent induction was shown, with 0.01 μM
Dex leading to low-level expression and 1 μM Dex for optimal induction. Localised expression of
the transgene at sites of induction, coupled with non-leakiness and tight regulation with plants
developing normally in the absence of induction, suggest that pOpOn2(Kan)::YFP:ICK1/KRP1109 is
a promising system for future studies in localised induction.
Keywords: leaf morphogenesis, plant cell division, segregation analysis, macroinduction, pOpOn2
transactivated overexpression system, dexamethasone, ICK1/KRP1
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Introduction
The precise role of cell division in leaf shape and size has been an unresolved debate. The general
concept of plant morphogenesis involving the formation of shape and structure by coordinated cell
division and growth, a fundamental question remains unanswered: “To what extent do oriented cell
divisions contribute to the determination of form?” (Haber 1962). Two polarized theories have
been proposed, the cellular theory suggests cell division as the driver of growth and development
whereas the organismal theory considers cell division simply follows a developmental plan
(reviewed in Fleming 2006; Inzé and De Veylder 2006). Experimental evidence largely supports an
organismal view (Beemster et al. 2003) in that leaf morphogenesis proceeded normally with
compensated cell enlargement (Tsukaya 2006) despite the suppression of cell division (Haber 1962;
Hemerly et al. 1995; but cf. Wang et al. 2000).
Increasing knowledge of the mechanistic basis of cell cycling has provided novel methods to
manipulate the spatial and temporal patterns of cell division in plant development. For example, the
modulation of cell division through localised microinduction by Wyrzykowska and colleagues
(2002). A transient increase of cell proliferation in primodia through transient expression of an Atype cyclin Nt;CycA3;2 and a phosphatase Sp;cdc25 surprisingly resulted in lamina indentation at
the site of induction whereas inhibition of cell division by roscovitine (a CDK inhibitor) led to
bulging. Although counter-intuitive, this evidently supports the influence of cell division in
morphogenesis.
Quantitative analysis of the cell division patterns during development provides a solid
foundation in the mechanistic understanding of leaf morphogenesis since the orientation of cell
division and growth are closely related (Fleming 2006). Visualisation of the planes of cell division
through aniline blue staining of callose on newly formed cell plates (Bougourd et al. 2000) allows
computational modelling based on the position and orientation of cell division. A dexamethasone
(Dex)-inducible transactivated expression system (Moore et al. 2006) is adopted to locally modulate
cell division. Perturbations at different stages of leaf development can be monitored through
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transient overexpression of ICK1/KRP1109, a truncated plant cycling-dependent kinase inhibitor
(CKI) protein lacking its negative regulatory domain. ICK1/KRP1109 protein acts non-cellautonomously in a concentration-dependent manner blocking entry into mitosis and promoting Sphase progression, results in endoreduplication (Weinl et al. 2005). However, normal cell fate
specification, despite the ICK1/KRP1-induced endoreduplication (Weinl et al. 2005) which is
usually associated with terminal differentiation e.g. trichomes (Sugimoto-Shirasu and Roberts 2003),
renders it a suitable candidate as an endogenous inhibitor of cell division.
The current project focused on the characterisation of four promising lines of transformed
Arabidopsis (T3 generation) with the primary goal to optimise the pOpOn2 overexpression system
(explained in Fig. 1). The first step is to select for a homozygous line with the highest level of gene
expression upon Dex-induction. This involved segregation analysis of a kanamycin-resistance
marker (Kanr) and the quantification of yellow fluorescent protein (YFP) expression through image
analysis. It is assumed that a homozygous transgenic line will show high expression of YFP (Moore
et al. 2006). In situ expression of YFP provides a strong evidence for the presence of its fused
counterpart, ICK1/KRP1109. Hence, selecting transgenic line with the greatest YFP expression could
enhance the required functionality of the fusion construct. Further trial studies, which employ a
macroinduction technique in an attempt to simulate microinduction, were carried out to identify the
optimum concentration of Dex application for localised induction with Sephadex® beads (Fleming
et al. 1997). Ideally, the inducible system has to be non-leaky and tightly regulated, which signify
its temporal and spatial control.
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- Dex
+Dex
Figure 1 Schematic diagram of the T-DNA construct.
Kanr expression cassette comprises of neomycin phosphotransferase II gene (NPTII) with constitutive nopaline
synthase (NOS) promoter which only becomes activated after Ti plasmid enters a plant cell. The Dex-inducible
pOpOn2 transactivating system contains two transcription units. The first unit employs a constitutive CaMV 35S
promoter to express a Dex-responsive chimaeric transcription factor (LhGR). The second unit consists of six copies
of the transcription factor binding site (artificial promoter „pOp‟) linked to truncated CaMV 35S minimal promoters
and TMV omega translation enhancers, which forms the birectional promoter (pOp6) used to express the fusion
construct and β-glucuronidase gene (GUS). The fusion construct of yellow fluorescent protein (EYFP-N1) and Nterminally truncated (deleted amino acids 2 to 108) KIP related protein (ICK1/KRP1 109), from Weinl et al. (2005),
was inserted into the GATEWAYTM cloning site. Each gene or construct ends with poly-A signal which serves as
transcription terminator. Circles represent the dexamethasone, a strong synthetic glucocorticoid (Aoyoma and Chua
1997), which binds and activates LhGR, in turns result in the transcription of both GUS and YFP:ICK1/KRP1109
(shown by the bidirectional arrow).
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Materials and Methods
Growing transgenic lines
Seeds used are from selfed T2 generation of transformed A. thaliana (ecotype Columbia) lines 22-4,
22-5, 22-6 and 22-8 with pOpOn2(Kan)::YFP:ICK1/KRP1109 (Weinl et al. 2005; Fig. 1). T-DNA
insertion in these four chosen transgenic lines was previously verified through PCR with positive
results in YFP expression and GUS staining.
For segregation analysis of kanamycin-resistance marker, seeds were surface-sterilised with
20% bleach in 0.05% Tween-20 for 10 min, rinsed thrice with autoclaved water, and stored for 4
days at 4oC devoid of light. Stratified seeds were then sown onto square Petri plates (12 x 12 cm, 6
x 5 seeds, 2 plates per line) containing 50 ml of growth medium with 0.5X MS salts (SigmaAldrich), 1% (w/v) sucrose, 0.8% (w/v) plant agar (Duchefa Biochemie) and 50 μg/ml kanamycin.
Plates were placed in a growth cabinet at 22oC day/ 20oC night under a 16 h photoperiod at light
intensity of 100 μmol m-2 s-1. Seedling development was monitored for kanamycin resistance (Kanr)
up to the eighth day. Segregation analysis study was repeated due to contamination of 0.1% agar
solution used in seed plating.
In the study of macroinduction using different concentrations of dexamethasone (Dex), seeds
of wild type Col-0 (as growth control), and line 22-4 (with the greatest number of Kanr seedlings)
and line 22-8 (with the most intense YFP expression) were sterilised and stratified as before. Ten
seeds of each line separated by ten seeds of Col-0 were sown onto the same square Petri plate
containing growth medium as above but without kanamycin to avoid any adverse physiological
effect of antibiotic. Each Petri plate was assigned to one Dex concentration treatment.
Induction of YFP expression – floating test
The fifth (control) and the sixth (+Dex) leaves of eighteen-day-old Kanr seedlings from the repeated
segregation study were harvested using aseptic forceps. Each leaf was placed floating in a well
containing either 300 μl of 10 μM Dex prepared from 10 mM stock (Sigma) in dimethyl sulfoxide
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(DMSO) through 1:1000 dilution with water, or 300 μl of 0.1% (v/v) DMSO (control). The 96-well
plates were then placed in growth cabinet for 24 h before examining the YFP expression.
YFP check and image analysis
YFP expression on the adaxial surface of the leaves were observed using fluorescent stereo
microscope Leica MZFL-III with GFP3 filter (470/40 nm exciter| 525/50 nm barrier) under 2.0X
objective. Monochromatic images were taken using SPOT RTKE741 slider colour/mono microscope
digital camera with the accompanying SPOTTM software (Diagnostic Instruments). Image capture
was standardised with settings at 2x2 binning, gain 4 and exposure time of 2 sec. Images were
saved as uncompressed .tiff files at 8 bits per pixel.
The images were then analysed using freeware Scion Image (Scion Corp). For each leaf, a
representative area was selected to measure the average greyscale value i.e. 0 (white) to 255 (black)
which corresponds to the brightness of the image, hence the fluorescence intensity of YFP.
Measurement files for each line were imported into spreadsheet for tabulation and analysis.
Macroinduction
Macroinduction was carried out on leaves of 1 to 1.5 mm in length (as compared to 200 μm in
microinduction) which are sufficiently early in leaf development that cell division is still active
(Donnelly et al. 1999). This is essential for the observation on any morphological effect of
transgene expression and for the modelling of cell division pattern.
Macroinduction was performed to simulate microinduction by using Sephadex® G-100
(Amersham Biosciences) beads (10-40 μm) mixed with desired concentration of Dex. To study the
use of different Dex concentrations, 1 μM, 100 nM and 10 nM Dex were prepared through
sequential 1:10 dilution of 100 μM frozen stock with Sephadex solution (500 mg/20 ml H2O, rinsed
thrice until pH neutral). Mixed solution was applied to the left flank of one leaf of each first pair in
eight-day-old seedlings (N=5 per line) using a glass Pasteur pipette with a fabricated capillary end.
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The untreated leaf of the first pair was used as the control in GUS assay. No DMSO control was
applied due to technical difficulty.
The study was repeated due to the leakiness during application in trial experiment. Application
of Dex was refined to improve induction procedure by preparing Dex solutions and Sephadex
solution separately, mixed on a glass slide prior to application. To verify earlier results, two of the
previously treated seedlings were chosen in addition to two untreated seedlings for each transgenic
line (22-4 & 22-8). Dex treatment was applied to the left flank of all leaves. All YFP analyses and
GUS assays were performed 24 h after induction.
GUS assays
Harvested leaves were placed in 96-well plates and vacuum infiltrated for 10 min with 100 mM
NaH2PO4, pH 7.5, containing 0.5 mg/ml 5-bromo-4-chloro-3-indoyl-β-D-glucuronide (X-GlcA)
prepared from frozen stock of 20 mg/ml X-GlcA (Apollo Scientific) dissolved in DMSO, 1 mM
K3Fe(CN)6, 1 mM K4Fe(CN)6, 0.1% (w/v) Triton X-100, 10 mM EDTA and incubated at 37°C in
the dark for 24 h. Tissue samples were then fixed in 70% ethanol. In the repeated study using
different Dex concentrations, the same procedure was followed but 12-well plates were used for
whole seedlings.
Nomarski microscopy
GUS stained tissue samples which were fixed overnight in 70% ethanol were cleared in 50%
economic bleach, and stored in Eppendorf tubes with 90% ethanol at room temperature. For
histological study, the samples were mounted on slides with a drop of water and examined under
Nomarski optics using an Olympus BX51 microscope. This allows detailed visualisation of tissue
structures across all layers conveniently without the need for histological sectioning or the use of
Hoyer's solution with toxic chloral hydrate (e.g. Bougourd et al. 2000).
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Results
Segregation analysis
All stratified seeds started to germinate two days after seed sowing on agar growth medium with the
emergence of root and cotyledons. Infestation by fungus in all Petri plates became evident on the
sixth day. However, the difference between the effects of fungus and kanamycin was
distinguishable by the retarded growth and redden leaves of seedlings near or in the fungus colonies
as compared to stunted development among seedlings which were kanamycin-sensitive. The
numbers of kanamycin-resistant (Kanr) seedlings were determined on day 8 after the emergence of
the first leaf pairs (~1 mm). A new batch of seeds were sown to confirm the results from previous
observations and to be used for further studies. All seeds were fully germinated with the appearance
of first leaf pairs by the end of day eight when kanamycin-sensitive seedlings became apparent.
In total, 486 seeds from four transgenic lines were sown and successfully germinated for the
screening of homozygous line. The observations were consistent between the two studies and the
results were pooled (Table 1). Line 22-4 was shown to be homozygous with respect to kanamycin
resistance marker whereas lines 22-5, 22-6 and 22-8 appeared to be heterozygous.
Table 1 Segregation analysis of four pOpOn(Kan)::YFP:ICK1/KRP1109 transformant lines. Seeds (T3
generation) were geminated on 0.5X GM plates containing 50μg/ml kanamycin and the number of resistant (R)
and sensitive (S) seedling was counted after 8 days. Results shown are pooled data from two independent
studies.
% Kan
Line
Resistant
Sensitive
st
1 study
nd
2
χ 2 test
r
study
Pooled
R:S ratio
χ2
22-4
123
0
100
100
100
1:0
-
22-5
85
35
72
70
71
3:1
1.11*
22-6
98
25
82
78
80
3:1
1.43*
22-8
94
26
80
77
78
3:1
0.71*
* P>0.05, Chi-square ( χ ) goodness of fit test (d.f.=1)
2
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YFP expression
In order to verify the expression of the fusion construct YFP:ICK1/KRP1109, all of the Kanr
seedlings from the repeated study of segregation analysis were tested for YFP expression. 0.1%
DMSO was used as a control to check the leakiness of the inducible system. None of the controls
(N=192) was induced but the site of incision on the petiole showed weak YFP expression (Fig. 2).
Mean greyscale values, 0 (white) to 255 (black), from image processing were used to represent
the expression of YFP, such that the greater the greyscale value, the lower the fluorescence intensity
(YFP intensity). Threshold (background) values for each Petri plate were determined by using the
average greyscale values calculated from respective controls (Fig. 2). Samples induced with Dex
10 μM (+Dex) which gave values greater than the threshold were considered to have no YFP
expression. Mean greyscale values for +Dex were then evaluated using only the induced samples
with YFP expression. There was no significant difference between samples from separate Petri
plates except for transgenic line 22-4 (2-sample t-test: T=2.22, d.f.=34, P=0.03). There was a
significant difference in the mean level of YFP expression among the transgenic lines (Fig. 3,
ANOVA: F3, 143=7.09, P<0.001). Mean YFP expression in line 22-8 was about twofold of that line
22-4, whereas lines 22-5 and 22-6 appeared to be intermediate.
Individual value of each leaf sample induced with Dex 10 μM is depicted in figure 4. There
was a distinct cut-off point in which YFP expression can be either very intense (<201) or variably
distributed (205-244). Therefore, the thresholds for identifying samples with no YFP expression
and to select for seedlings with great level of expression in the combined data were decided to be
245 and 200 respectively. A total of eight seedlings with the highest level of YFP expression were
transferred to soil to be selfed for the T4 generation.
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Left
Line
Right
+ Dex
Control
24
a
+ Dex
31
b
Control
24
31
22-4
230.4 ± 2.1
246.2 ± 0.1
10
c
235.6 ± 1.1
14
d
246.1 ± 0.1
19
23
22-5
227.1 ± 4.2
246.4 ± 0.1
18
e
225.8.4 ± 2.2
24
f
246.3 ± 0.1
18
24
22-6
221.3 ± 2.8
247.8 ± 0.2
20
g
226.7 ± 3.0
22
h
246.8 ± 0.1
14
23
22-8
220.9 ± 3.6
246.5 ± 0.1
225.1 ± 2.5
246.6 ± 0.1
Figure 2 Floating test results showing the induced YFP expression of leaf 6 with Dex 10 μM and the control leaf 5
with 0.1% DMSO among the 18-day-old seedlings. Samples of the most intense fluorescence intensity of the left and
right Petri plates (independent samples) with their respective controls from line 22-4 (a, b), line 22-5 (c, d), line 22-6 (e,
f) and line 22-8 (g, h). Values on the bottom left corner are the mean greyscale values ± 1 SE calculated using only the
samples with YFP expression. For the controls, these values represent the thresholds in determining YFP expression for
+Dex. Number on the top right corner is the sample size (N). Scale bar = 400 μm.
Relative mean YFP intensity
30
b
25
b
ab
20
15
a
10
5
0
22-4
22-5
22-6
22-8
Line
Figure 3 Mean relative intensity of YFP expression for
the four transgenic lines in the floating test. Relative
value of YFP intensity was calculated from the difference
in the greyscale value of induced leaf samples with
threshold value of 245. Error bars are 1 SE. Alphabets
represent results from Tukey multiple comparison test
(P>0.05) in which line 22-4 is significantly different
from lines 22-6 and 22-8 but not line 22-5.
Figure 4 Individual value plot showing greyscale values
of all four lines from the floating test. The reference lines
indicate the thresholds for choosing seedlings to be
propagated (filled dots) or to determine the absence of YFP
expression (crosses). Boxplot only includes sample with
YFP expression. The boxes indicate the interquartile range.
The horizontal lines indicate the median, the squares
represent the mean. The whiskers extend to the highest or
lowest value within 1.5 times the interquartile range.
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40
22-4
22-5
22-6
22-8
35
Proportion (%)
30
25
20
15
10
5
0
-189
-199
-209
-219
-229
-239
-245
>245
Greyscale value
Figure 5 Proportion of each class of greyscale value showing the distribution of YFP expression for each transgenic
line. X-scale values are the upper class limits. >245 include all samples with mean greyscale value over 245 which
were considered to have no YFP expression.
The proportions of induced seedlings with no YFP expression were similar across all four
transgenic lines (Fig. 5) and were statistically significant in following 3:1 ratios (Table 2) as expected
for heterozygous lines. Furthermore, lines 22-4 and 22-5 appeared to have similar patterns of YFP
expression as compared to lines 22-6 and 22-8 (Fig. 4 & Fig. 5).
Table 2 Floating test results of Kanr seedlings showing induced
expression of YFP and failed induction follows a 3:1 ratio.
χ 2 test
YFP expression
Line
Presence
Absence
ratio
χ2
22-4
48
14
3:1
0.19*
22-5
29
8
3:1
0.23*
22-6
36
12
3:1
0*
22-8
34
11
3:1
0.01*
* P>0.05, Chi-square ( χ ) goodness of fit test (d.f.=1)
2
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Macroinduction
Results above showed that line 22-4 and line 22-8 were different in the number of Kanr seedlings
and the induced expression of YFP. However, early seedling growth and development were
observed to be uniform between the two transgenic lines and were comparable to the wild-type
plants. To find out which line is a better candidate for microinduction, macroinduction experiments
were carried out on lines 22-4 and 22-8. Different Dex concentrations were used to study the
optimum concentration.
The first trial was performed on the first leaf pairs of eight-day-old seedlings. Five seedlings
from each line were used for each Dex concentration. Results (Table 3) showed that line 22-4 was
more likely to be induced than line 22-8. All three Dex concentrations of 1 μM, 100 nM and 10 nM
were able to induce transgene expression. However, there was a discrepancy between the results
from YFP analysis and GUS staining. In all cases, YFP analysis showed less than half of the
positive result as compared to GUS staining. Up to half of the controls were induced by the leakage
of Dex at high concentration, partly due to the small leaf size (1 mm) compounded by the wet
adaxial epidermal surface of the newly-emerge first leaf pairs.
Table 3 Summary results from the trial macroinduction on the first leaf pairs of eightday-old d seedlings (N=5 per line). The untreated leaf of the first pair was used as the
control in GUS assay.
Proportion of positive results
Dex
concentration
1 μM
100 nM
10 nM
Line
YFP signal
22-4
22-8
Combined
0.6
0.2
22-4
22-8
Combined
0.2
0
22-4
22-8
0.2
0.4
Combined
0.4
GUS assay
+Dex
Control
1
0.6
0.8
0.8
0.2
1
0.4
0.4
0.2
0.1
0.7
0.6
0.6
0.3
0.5
0.3
0
0
0.6
0
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In the repeated study, induction was
including
leaf
number
(related
to
the
developmental stages) as an additional factor of
investigation. The results (Table 4) confirmed
that a Dex concentration of 10 nm was
sufficient to induce the transgene expression
with similar success rate as 100 nM and 1 μM
Proportion of positive results
carried out on leaves of various sizes by
0.80
YFP
0.70
GUS
0.60
0.50
0.40
0.30
0.20
0.10
0.00
1 μM
(Fig. 6). Successful induction in line 22-8 was
greater, contrary to that observed in first trial.
100 nM
10 nM
Dex concentration
Figure 6 The proportion of successful inductions with
different concentrations of Dex as indicated by the YFP
The technical improvement was evident with
the localised induction on the left flank of the
expression and GUS staining in combined data of line
22-4 and line 22-8 (N=8).
leaves except for leaf 7 which appeared to be
localised to the apical regions (Fig. 7).
Table 4 Summary results from the repeated study on Dex concentrations on
eighteen-day seedlings (N=4 per line). Positive results indicate the evidence of
induction on at least one of the treated leaves on each seedling.
Dex
concentration
1 μM
100 nM
10 nM
Proportion of positive results
Line
YFP signal
22-4
22-8
Combined
0.75
0.25
22-4
22-8
Combined
0.50
1
22-4
22-8
0.25
1
Combined
GUS assay
0.50
1
0.50
0.75
0.75
0.75
0.75
0.75
0.25
1
0.63
0.63
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4
1
7
6
2
3
5
a
b
c
d
e
f
Figure 7
Observations from macroinduction.
GUS staining on the 16-day-old seedlings from line 22-8 after macroinduction with Dex 1 μM (a), 100 nM (b) and 10
nM (c). Note the GUS staining of leaf 7 was localised to the apical regions. (d-f) Localised YFP expression on leaf 5
(marked by white arrowheads in (a-c)) at the site of macroinduction for each corresponding Dex concentration (a:d, b:e
and c:f). Black arrowheads are pointing the sites of induction. Numbers represent the leaf number (1 and 2 are the first
leaf pair, and 3 to 7 are sequentially younger leaves). Scale bars = 1 mm (a-c); 500 μm (d-f).
staining and YFP expression appeared to
correlate with the concentration of Dex (Fig. 7).
For 16-day-old seedlings, leaf 5 was most
successfully induced (Fig. 8) but the result is
unlikely to be statistically significant due to low
sample size (N=8). At Dex concentration of 1
μM, the induction on leaf 3 of line 22-8 was
Proportion of successful induction
The intensity of induction as seen on the GUS
0.7
1 μM
100 nM
0.6
10 nM
0.5
0.4
0.3
0.2
0.1
0
more intense than line 22-4 (Fig. 9). Trichomes
were distinctively stained in both leaves.
Diffuse patterns of induction were observed in
7
6
5
4
3
Leaf number
Figure 8 The proportion of successful induction with YFP
expression in leaves 3 to 7 (refer Fig. 7) of the 16-day-old
seedlings (N=8) from lines 22-4 and 22-8 at different
leaf 3 with GUS staining on guard cells,
concentrations of Dex.
palisade mesophyll and spongy mesophyll cells
but absent in pavement cells (Fig. 10 e & f).
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a
b
c
d
e
Figure 9
f
Analysis of localised induction.
Leaf 3 of 16-day-old seedling from line 22-8 (refer figure 8a) (a,b) and line 22-4 (c-f) induced with Dex 1 μM
showing different intensity of GUS staining. (e,f) Nomarski views under Olympus BX51 microscope showing diffuse
pattern of GUS staining in guard cells, palisade mesophyll and spongy mesophyll cells. Note indentation in (c)
indicated by the arrow was a result of non-intentional induction early in development from leakage in first trial. Scale
bars = 500 μm (a,c); 100 μm (b); 50 μm (d); 20 μm (e,f).
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Discussion
To test models of plant development, high consistency in the tools used to manipulate growth is
desirable to ensure any observed effects are not artefacts reflecting large range of variation among
individuals but are the results from a given treatment (Moore et al. 2006). In transgenic plants, this
means finding homozygous lines to minimise the genetic variation within the samples and to
optimise the reproducibility of the study. From the segregation analysis, line 22-4 was identified as
the only transgenic line homozygous with respect to the kanamycin-resistance marker. However,
the induced-expression of YFP in kanamycin-resistant seedlings of all T3 transgenic lines followed a
3:1 ratio. Furthermore, line 22-4 appeared to have a significantly lower intensity of YFP expression,
contradictory to that of expected for a homozygous line. Further evaluation on choosing the best
line for microinduction was carried out through macroinduction studies on line 22-4 and line 22-8
by testing the outcome of different Dex concentrations.10 nM of Dex was shown to be adequate to
induce low-level transgene expression. However, successful inductions were variable for both
transgenic lines with line 22-8 showing more intense induction than line 22-4. Leaf 5 of the 16-dayold seedlings appeared to be the best candidate for the macroinduction study using Sephadex®.
Possible scenarios of the variable expression of transgene
The simplest explanation for the homozygosity of transgenic line 22-4 in Kanr marker but
heterozygous with respect to YFP marker (fusion construct) is that the two markers follow the
Mendelian principle of independent segregation in T2 generation. Kanr and YFP markers are located
at the extreme ends of a considerably large T-DNA construct (Fig. 1) such that, it is possible for one
of the inserted constructs to lose the fusion construct resulting in Kanr/- (Fig. 10). If line 22-4 has
two insertions, T3 generation would be 100% Kanr but only 75% with YFP expression which is
consistent to that observed. Furthermore, presence of recombination hotspots within CaMV 35S
(Ho et al. 2000) used in the T-DNA (Fig. 1) supports the separation of Kanr marker from the rest of
the construct. The same is applicable to other transgenic lines such that recombination events result
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in a separation mosaic of the T-DNA constructs (Fig. 10). Consequently, only three-quarter (9:3) of
the 75% (12:4) Kanr seedlings had YFP expression. However, this cannot explain the YFP
expression pattern (Fig. 4) of much fewer seedlings with intense transgene expression than expected
if independent segregation and recombination hold true, assuming multiple insertions showing
greater YFP expression than single insertion.
On the other hand, this could be a common phenomenon of variable transgene expression
(Matzke and Matzke 1998). Firstly, it could be due to the position effects such that a transgene
insertion near enhancer elements or actively transcribed regions of the genome is more likely to be
expressed. Hence, by chance a few seedlings with inserts near to highly expressed region might
give rise to the observed intense expression of YFP, and those fall into regions where gene
expression is repressed result in silencing. Otherwise, it could be a consequence of genomic
imprinting common for homozygotes in the early stages of embryogenesis (Moore et al. 2006) such
that one of the inserts in line 22-4 (presumable homozygous) was silenced resulting in a much lower
expression of YFP in the floating test compared to other heterozygous lines.
Kanr /
YFP
Kanr
/-
-/
YFP
-/-
Kanr /
YFP
**
*
**
*
Kanr
/-
*
O
*
O
-/
YFP
**
*
X
X
-/-
*
O
X
X
* Expression of YFP (** greater intensity)
O Kanamycin-resistant without YFP expression
X Kanamycin-sensitive
[-/-] Absence of transgene
Figure 10 Possible combinations of transgene composition with independent segregation and recombination of Kanr
and YFP markers. Yellow and red boxes represent the expected phenotypes of a heterozygous line. Blue boxes are
proposed products of recombination events. Underlined symbols represent the scenario for transgenic line 22-4.
17
050138401
Thirdly, repetitive sequences are often subjected to silencing potentially as a defence response
against foreign DNA (Matzke and Matzke 1998). This is possible for pOp6 promoter which consists
of six repeated units of pOp. Silencing of the pOp6 bidirectional promoter will result in silencing of
GUS and the fusion construct, similar to the effect of losing part of the insert. However, a regular
pattern of silencing on only one of the insertions is unlikely. Previous studies using the pOp/LhGR
Dex-inducible system (Craft et al. 2005) demonstrated that pOp6 promoters remain active in most
lines even after eight generations. In the cases of silencing in which the induced phenotype has been
lost, inducible GUS expression from the same pOp6 operator array was still active. This suggests
the silencing occurred through sequences outside the operator array as a characteristic of
Arabidopsis sequences rather than the expression system (Moore et al. 2006).
The most effective way to test these possibilities is to determine the number of inserts through
Southern blotting and verification by PCR amplification of the inserted marker gene. If independent
segregation or recombination is the case, there will be fragments of truncated T-DNA whereas
complete sequences may indicate silencing effects on the transgene. Otherwise, it could also due to
post-transcriptional gene silencing (PTGS). Sequence-specific PTGS has been proposed by
Schubert et al. (2004) such that individual transgenes have a specific threshold of mRNA
abundance that will trigger PTGS. Greater abundance of a transgene is more likely to be silenced.
Parallel to the assumption on the homozygosity of line 22-4 with two insertions, PTGS might be
responsible for the lower observed YFP expression - yet to be verified through RT-qPCR.
Furthermore, there could even be a link to the microRNA-mediated regulation of gene expression as
observed in the control of leaf morphogenesis (Palatnik et al. 2003). Hence, the success of induction
can be determined by the fusion construct as much as the expression system itself. The 3:1 ratios
could just be the inherent chance of the transactivating system to be functional 75% of the times
following induction with Dex. These possibilities need not be mutually exclusive.
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050138401
Functionality of the overexpression system
There is still no study at present that characterises the pOpOn2 overexpression system (Fig. 1)
which was originally derived from the successfully applied inducible RNAi pOpOff2 system
(Moore et al. 2006; Wieloposka et al. 2005) that utilises the GatewayTM cloning site for the rapid
production of hpRNA constructs. A fusion construct of YFP and N-terminally truncated
ICK1/KRP1 lacking the negative regulatory domain has been shown previously to produce a fully
functional protein (37 kD) found in both nucleus and cytoplasm (Weinl et al. 2005). Therefore, it is
reasonable to assume that the fluorescence intensity of YFP will correlate to the intended activity of
the cyclin-dependent kinase inhibitor in blocking mitosis. Furthermore, the non-intentional
induction during an early leaf stage resulted in indentation on the leaf flank (Fig. 9). This is
consistent with the serrated leaf shape from 35S::ICK1/KRP1 overexpression (Wang et al. 2000),
suggesting that the fusion protein is functional.
Despite the discrepancies seen in kanamycin resistance and the YFP expression, it is clear that
this inducible overexpression system is non-leaky and tightly regulated. There were no observed
lethal effects with all transgenic lines fully germinated and developed normally in the absence of
inducer. All of the controls in floating test were consistent in showing no sign of YFP expression
except at some of the wounded petiole. This indicates the potential effects of DMSO 0.1% or
wounding in causing transgene expression but unlikely to be significant in jeopardising
microinduction since expression is localised to wounded site.
Non-leakiness of the system is further supported by the tightly localised induction in the
macroinduction studies despite ICK1/KRP1 previously found to move between cells (Weinl et al.
2005). It is shown that the transgene expression is concentration-dependent ranging from 0.01 to 10
μM but no quantitative data are available. This is consistent with the dose-dependency nature of the
Dex-inducible expression system (Aoyoma and Chua 1997) with full induction potentially
achievable by 1 μM dexamethasone and it showed that a Dex concentration as low as 10 nM was
sufficient for modest transgene induction.
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050138401
However, all data obtained from the macroinduction trials need to be interpreted with caution
due to the small sample size (N=4/5) and the lack of DMSO controls. These preliminary
observations from the macroinduction experiments were meant to be suggestive with different
factors investigated to generate hypotheses for more detailed studies. For example, concentrations
of Dex and the localised induction on different leaf number need to be verified independently.
Although most results from GUS assay were comparable to that of YFP analysis, it appeared
that there were disparities between the two tests in verifying transgene induction with increasing
concentrations of Dex. GUS staining had been observed on leaves without YFP expression. This
could due to the time lag between the two tests or a greater sensitivity of GUS assays but strong
inference cannot be made due to variable results obtained by using non-stable transgenic lines in the
investigation.
Implications for future studies
Trial studies of macroinduction reflect the technical challenges in this field for leaves smaller than
200 μm. Sephadex® was chosen because individual beads are suitable in providing a small (10-40
μm) localised source of inducer. The apparent leakiness of the inducer solution can be minimised by
selecting single beads. However, even under the stereo microscope with high magnification, greater
precision of induction will still be challenging.
Proposed correlation between the dose-dependency of Dex induction strongly suggests its
applications in the investigation of the concentration-dependent role of ICK1/KRP1 (Weinl et al.
2005). Gene expression levels can be modulated using different concentrations of Dex. This allows
both weak and strong phenotypes to be examined in the same plant line. Furthermore, different cell
types should be considered as suggested by more induced palisade cells than epidermal cells at the
site of induction (Fig. 9), such that physiologically active mesophyll cells have greater gene
expression (similarly in guard cells and trichomes) (Donnelly et al. 1999).
The selection for homozygous line purely based on kanamycin-resistance was shown to be
insufficient as reflected by the direct observations from the expression of YFP fusion construct.
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050138401
Selecting for a homozygous line might not be the best option taking gene silencing into
consideration. Hence, for greater confidence when choosing for best performing transgenic line, one
should vigorously assess the ultimate gene expression and the functionality of the expressed protein.
YFP fusion protein has shown to greatly facilitate such purpose and eases Dex concentration assays
for future characterisation of the relationship between Dex concentrations and induction levels.
Variable results obtained from the macroinduction studies together with the apparent variation
in the pattern of trichome distribution even within the same transgenic line (Fig. 8) reiterate the
need for a stable transgenic line with consistent expression of transgene apart from the enrichment
of high-level expression. The variability in the transgenic lines is hoped to be circumvented using
the progenies of the eight best performing seedlings chosen for propagation. In conclusion, this
project has demonstrated that pOpOn2(Kan)::YFP:ICK1/KRP1109 is a promising system which is
non-leaky and useful for localised induction studies.
Acknowledgements
I am grateful to Asuka Kuwabara who had patiently taught me all the technical skills and stimulated
me to think critically. I want to thank Marion Bauch, Robert Malinowski and other colleagues who
have welcomed me to their laboratory. Last but not least is to thank Andrew Fleming as my project
supervisor for the enlightening discussions we had and his helpful advice throughout the project.
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