Caged gene-inducer spatially and temporally controls gene expression and plant development in transgenic Arabidopsis plant

Article abstract of DOI:10.1016/j.bmcl.2006.01.103

Two new types of caged gene-inducers, caged 17β-estradiol and caged dexamethazone, were synthesized. Caged gene-inducers were applied to transgenic Arabidopsis plants carrying a steroid hormone-inducible transactivation system. Light uncaged caged gene-in

Full text of DOI:10.1016/j.bmcl.2006.01.103

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2470–2474
Caged gene-inducer spatially and temporally controls
gene expression and plant development in
transgenic Arabidopsis plant
Ken-ichiro Hayashi,a,* Kazuya Hashimoto,a Naoyuki Kusaka,a Atsushi Yamazoe,a
Hidehiro Fukaki,b Masao Tasakab and Hiroshi Nozakia,*
aDepartment of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama City 700-0005, Japan
bGraduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, 630-0101 Ikoma, Nara, Japan
Received 21 September 2005; revised 20 January 2006; accepted 23 January 2006
Available online 9 February 2006
Abstract—Two new types of caged gene-inducers, caged 17b-estradiol and caged dexamethazone, were synthesized. Caged gene-in-
ducers were applied to transgenic Arabidopsis plants carrying a steroid hormone-inducible transactivation system. Light uncaged
caged gene-inducers and controlled spatial and temporal expression of transgene in the transgenic plant. Furthermore, caged
gene-inducers enabled the control of root development by light.
ꢀ 2006 Elsevier Ltd. All rights reserved.
In multicellular organisms, the expression of individual
genes is temporally and spatially regulated to maintain
proper biological processes. The control of specific gene
expression in transgenic organism is a powerful technol-
ogy to assess the biological function of specific proteins,
which have greatly contributed to biology. Chemical-in-
ducible gene expression systems have been widely used
to control temporal expression of the transgene by
chemicals.1,2 The spatial control of gene expression
was partially achieved using tissue-specific promoter to
activate the transgene expression.3–5 However, the reso-
lution of the spatial control and the expression level
depend on the nature of each native promoter.
by photo-irradiation and allowed the temporal and
spatial control of specific protein expression, because
light is easily controllable in terms of time, area, and
intensity. However, these approaches require complicat-
ed experimental manipulations, such as caging the target
molecules and incorporating them into cells, in order to
study the protein of interest.
An alternative approach is the combination of caged
chemical-inducer and chemical-inducible gene expression
system, namely a caged chemical gene-inducer system. In
transgenic cell harboring a chemical-inducible transgene,
a caged chemical-inducer like caged steroid hormone can
be uncaged by a light irradiation and the uncaged inducer
can then drive transgene expression within the irradiated
area. Previous studies demonstrated that caged estrogen
agonist/antagonists,9,10 caged b-ecdyson,11 and caged
thyroid hormone agonists12 can control the reporter gene
expression in mammal cell culture upon their uncaging by
light. These studies demonstrate the feasibility of the sys-
tem in transiently transfected mammal cell cultures.
However, there is no report on the spatial and temporal
control of gene expression at the level of individual organ-
isms. In such caged chemical-inducer systems, the design
of the caged inducer is crucial for the control of spatial
and temporal gene expression, because the chemical and
biological properties, such as solubility, stability inside
cell, and cell permeability, depend on the nature of caged
molecules.
Caged compounds are inactivated bioactive molecules
with the functional group blocked by a photo-remov-
able protecting group (caging group). The original bio-
logical activity of a caged compound can be readily
recovered by photo-irradiation (normally 350–360 nm,
ultraviolet light). In previous studies, caged macromole-
cules related to transcription machinery, such as caged
DNA,6 caged mRNA,7 and caged GAL4/VP16 (transac-
tivator protein),8 were developed as a direct approach to
control the temporal and spatial expression of specific
protein. These caged macromolecules were activated
Keywords: Caged hormone; Gene expression; Light.
*
Corresponding authors. Tel.: +81 86 256 9661; fax: +81 86 256 9559
(K.H.); e-mail: hayashi@dbc.ous.ac.jp
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.103

K. Hayashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2470–2474
2471
We have designed new caged steroid hormones, caged
17-b-estradiol and caged dexamethazone, as caged
gene-inducers with carbonate linker and applied them
to transgenic Arabidopsis plants carrying two types of
chemical-inducible transactivation system. Arabidopsis
plants are ideal transgenic organisms for caged gene-in-
ducer system, since UV light could easily penetrate plant
tissues and two chemical-inducible systems have been
widely used for the molecular biological studies in Ara-
bidopsis. Furthermore, in contrast to mammal cell, ste-
roid hormones do not appear to affect plant growth.
for 2; dH 4.54 for 1, H-22 dH 4.51 for DEX; dH 4.86
for 3) attributable to carbonate linkage in 1H NMR
spectrum. Previously reported caged ER (2) was synthe-
sized according to the literature.9
To confirm the release of original hormones from caged
hormones 1 and 3 by UV light in vitro, UV-irradiated
solutions of 1 or 3 were analyzed by HPLC.15 After
1-min irradiation by a 6 W-UV lamp, the release of
ER and DEX was detected. A time course study indicat-
ed that UV-irradiation caused the reduction of caged
steroids and consequently released the original steroids
in a time-dependent manner. After 40-min exposure,
the yields for the release of original steroids from the
caged compounds were 45% for the caged ER (1) and
56% for the caged DEX (3), respectively. The quantum
yield (/reactant) of caged ER (1) and DEX (3) was deter-
mined to be 0.19 and 0.16, respectively.15 In addition,
caged steroids 1 and 3 were stable in dark after 24-h
incubation in a medium at 20 lM (data not shown).
These results indicate that caged steroids were uncaged
by UV light and the original hormones were released
in a light-intense-dependent manner, suggesting that
the release of hormones could be controlled by light
intensity (Fig. 1).
In this letter, we describe the spatial and temporal con-
trol of gene expression by light at individual organism
level and demonstrate light-regulated developmental
process in transgenic Arabidopsis plant.
To cage the 17-b-estradiol (ER) or dexamethazone
(DEX) with 1-(2-nitrophenyl)ethyl carbonate group,
we used the 1-(2-nitrophenyl)ethylcarbonyl imidazole
(4). This activated caging reagent is easy to be synthe-
sized and handled in comparison with a conventional
chlorocarbonate-caging reagent, because toxic phosgene
is usually used for the synthesis of chlorocarbonate from
the corresponding alcohol. 2-Nitrophenylacetophenone
was reduced with NaBH4 to yield a 1-(2-nitrophen-
yl)ethanol. This alcohol was reacted with 1,10-carbon-
yldiimidazole to give a caging reagent (4).13 This
activated caging reagent was mixed with cesium carbon-
ate and ER or DEX at room temperature for an hour to
afford a caged ER (1) or caged DEX (3), respectively.14
Caged steroids (1) and (3) were a mixture of C-70 epi-
mers in caging moiety. We used caged steroids as a mix-
ture of epimers for further studies because the two
epimers could not be separated even by HPLC. The po-
sition of 2-nitrophenylethyl carbonate was confirmed to
be C-17 position of ER and C-22 position of DEX by
the HMBC correlations (H-17/C-90 carbonyl carbon in
1 and H-22/C-90 carbonyl carbon in 3) and downfield
shifts of proton signals (H-17 dH 3.71 for ER; dH 3.73
To assess the spatial and temporal control of gene
expression by light in vivo, we applied caged ERs 1
and 2 to transgenic Arabidopsis pER8::GFP line.1 This
transgenic line strongly expresses a GFP reporter gene
under the control of the estrogen-inducible XVE trans-
activation system.1 In this system, the expression of the
transgene can be tightly regulated by estrogen in a dose-
dependent manner. Six-day-old pER8::GFP plants were
immersed in a medium containing 20 lM caged ERs 1
or 2 for 30 min and seedlings were then washed three
times with a medium lacking caged ERs. The irradia-
tion of UV light was carried out with a fluorescent
microscope.16 GFP expression could not be observed
without light irradiation (Figs. 2B and C, 1st panel).
By contrast, uniform GFP fluorescence was observed
in the whole root when light was irradiated over the
root after treatment with 1 or 2 (Figs. 2B and C, 2nd
panel). UV light was then irradiated as a spot in the
middle or the end region of a root (Figs. 2A–C). Figure
2C shows that spot illumination on a root failed to elic-
it spatial control of GFP expression, when transgenic
plants were treated with previously reported caged ER
(2). GFP fluorescence was uniformly observed over
the whole root, even when light was irradiated as a
spot. This might be due to the transport of ER from
2 that was bounded to the cell wall after washing.
Namely, 2 could not be washed out from the cell wall
after loading of 2 into cells. In addition to the photol-
ysis of intracellular 2, the absorbed 2 on the cell wall
was also uncaged by light. The released ER outside cell
would then diffuse through the entire root via transport
system such as the vessel and sieve tube. In contrast to
2, spot illumination after the incorporation of our
caged ER (1) induced spatial GFP expression at only
the irradiated area of a root (Fig. 2B), suggesting 1
can be used to control in vivo temporal and spatial
expression of transgene by light.
3'
O N
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18
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O
9'
5'
7'
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1'
11
10
17
O
1
4
H
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15
8
H
H
OH
HO
H
H
H
O
O
O
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O
18
OH
1'
9'
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11
22
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K. Hayashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2470–2474
glucocorticoid receptor binds with heat shock proteins
(HSPs) through its hormone binding domain located
at the carboxy-terminus of glucocorticoid receptor
(GR) to form an inactive heterodimeric complex in cyto-
plasm.17 The steroid hormone, DEX, dissociates HSPs
from the heterodimeric complex and allows the gluco-
corticoid receptor anchored in the cytosol to translocate
into the nucleus. Similarly, the fusion protein of a tran-
scription factor and GR is also inactivated by its
interaction with HSPs in the absence of steroid and
can be activated in the presence of steroid hormone.18,19
This GR-fusion system has been shown to work in
plants and has been widely applied to studies for
functions of plant transcription factor.20,21 Arabidopsis
pIAA14::GFP-mIAA14-GR line expresses the transla-
tionally fused protein, a mutated IAA14 fused with
GFP and GR at the N- and C-termini, respectively, un-
der the control of pIAA14 promoter.22 IAA14/SLR is
primary auxin-responsive gene that encodes short-lived
nuclear repressor regulating the development of lateral
root and root hair.23 Gain-of-function mutation of
IAA14/SLR confers the resistance of repressor protein
to degradation via the ubiquitin–proteasome pathway
and consequently the development of root hair and lat-
eral root is repressed in the mutant.23 Previous report
demonstrated that the treatment of this line with DEX
causes the accumulation of the fusion protein in
nuclei and blocked root hair development as shown in
Figure 3B.22 In wild-type plants, treatment with DEX
or 3 with/without light did not affect root hair formation
(Fig. 3A).16 In the transgenic line, treatment of caged
DEX (3) without light irradiation showed the same root
hair formation as observed in control (Fig. 3B). By con-
trast, no root hair was formed in the presence of 3 when
the root was irradiated by UV light (Fig. 3B). In addi-
tion, the UV-light irradiation enhanced GFP fluores-
Figure 1. Light uncages caged gene-inducers in a time-dependent
manner. Solutions of caged compound were irradiated by UV light and
released hormones were analyzed by HPLC. (A) Closed square, caged
ER (1); closed circle, ER. (B) Closed square, caged DEX (3); closed
circle, DEX. Relative amounts (%) for ER and DEX indicate released
rates from the original caged compounds.
We have applied caged dexamethazone (DEX) to anoth-
er chemical-inducible gene expression system in vivo.
In the absence of an appropriate steroid hormone, the
Figure 2. Spatial and temporal control of GFP reporter gene expression by light in root of Arabidopsis pER8::GFP plant. (A) UV light was
irradiated as a spot on roots by fluorescent microscopy for 3 s. Bar represents 1 mm. ER (1 lM) was used as a positive control. (B and C) Light-
activated GFP expression by caged ER (1) or previously reported caged ER (2). In the second panel, UV light was irradiated on the whole root. In
the third and fourth panels, UV light was irradiated as a spot. Arrow indicates the irradiated point. Bar represents 1 mm.

K. Hayashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2470–2474
2473
3. Brand, A. H.; Perrimon, N. Development 1993, 118, 401.
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14. Synthesis of 1 and 3. 17b-Estradiol (50 mg, 0.18 mmol) or
dexamethazone (50 mg, 0.13 mmol), CsCO3 (2 equiv), and
4 (1 equiv) were dissolved in THF and stirred for an hour
at room temperature in darkness. The reaction mixture
was extracted with EtOAc after the addition of brine. The
organic layer was evaporated. The mixture was subjected
to silica gel column and eluted with mixture solvents
(CHCl3/acetone 6:1 for 1 and CHCl3/acetone 4:1 for 3) to
yield epimeric mixture of 1 as a light yellow powder
(62 mg, 72%) and epimeric mixture of 3 as light yellow
powder (54 mg, 73%).
Compound 1 (70-epimeric mixture): UV kmax (MeOH)
nm (log e): 202 (4.47), 257 (3.56); IR (KBr) Vmax 3400,
Figure 3. Developmental control of root hair by light in
pIAA14::GFP-mIAA14-GR Arabidopsis transgenic plants. (A and B)
Wild-type (Col) or transgenic seedlings were treated with 0 or 50 lM of
caged DEX (3) for 30 min. DEX (10 lM) was used as a positive
control. UV light was irradiated on the indicated part for 3 s. (C)
Accumulation of mIAA14 repressor fused with GFP and GR in nuclei
by light. The root tip of the seedling was irradiated by UV light for 3 s.
DEX (10 lM) was used as a positive control.
1
2947, 1750 cmꢀ1; H NMR (CDCl3): d 0.80/0.81 (3 H, s,
CH3-18), 1.18–2.32 (13H, m), 1.69/1.71 (3H, d, J = 6.5,
CH3-80), 2.49 (2H, m), 4.54 (1H, t, J = 7.5 Hz, H-17),
4.85 (1H, br s, OH), 6.22 (1H, q, J = 6.5 Hz, H-70), 6.55
(1H, s, H-4), 6.61 (1H, d, J = 8.7 Hz, H-2), 7.12 (1H, d,
J = 8.7 Hz, H-1), 7.46 (1H, dd, J = 7.2, 7.1 Hz, H-40),
7.67 (1H, dd, J = 7.1, 8.0 Hz, H-50), 7.72 (1H, d,
J = 8.0 Hz, H-60), 7.98 (1H, d, J = 7.2 Hz, H-30); 13C
NMR (CDCl3): d 11.90/11.97, 22.05/22.17, 23.01/23.03,
26.08/26.11, 27.05, 27.22/27.30, 29.51, 36.71/36.78, 38.47,
42.99/43.02, 43.65, 49.53, 71.59, 86.53/86.60 (C-17),
112.66, 115.20, 124.52/124.55, 126.50/125.54, 126.91/
127.0, 128.47/128.50, 132.43, 133.78/133.78, 137.97,
138.13, 147.48/147.51, 153.31, 154.13; HRFABMS m/z
488.2029 (calcd for C27H31O6Na, 488.2049).
cence in nuclei (Fig. 3C).16 These observations indicate 3
was uncaged in vivo by light leading to the control of
root developmental process by activating the GR-fused
repressor.
Here, we demonstrate the spatial and temporal control
of gene expression by light at the organism level. This
study also shows that light-activated caged gene-induc-
ers can be used to control developmental process in Ara-
bidopsis plants. Our caged gene-inducers are potentially
powerful tools to assess protein functions at specific cells
and at specific developmental stage in plants.
Compound 3 (70-epimeric mixture): UV kmax (MeOH)
nm (log e): 205 (4.08), 240 (4.06); IR (KBr) Vmax 3200,
2932, 1743 cmꢀ1 1H NMR (CDCl3): d 0.84/1.03 (3H, s,
;
CH3-18), 0.87/0.89 (3H, d, J = 7.3 Hz, CH3-20), 1.21
(1H, m), 1.53/1.54 (3H, s, CH3-19), 1.55 (2H, m), 1.71/
1.72 (3H, d, J = 6.4 Hz, CH3-80), 1.72 (1H, m), 1.78
(1H, m), 2.05 (1H, m), 2.32 (3H, m), 2.58 (1H, m), 3.05/
3.08, (1H, m, H-16), 4.19 (1H, br s, H-11), 4.86 (2H, s,
H-22), 6.08 (1H, s, H-4), 6.23, (1H, q, J = 6.4 Hz, H-70),
6.31 (1H, d, J = 10.0 Hz, H-2), 7.21 (1H, d, J = 10.0 Hz,
H-1), 7.46 (1H, dd, J = 7.2, 7.3 Hz, H-40), 7.71 (1H, dd,
J = 7.2, 7.3 Hz, H-50), 7.82 (1H, dd, J = 7.3 Hz, H-60),
7.95 (1H, dd, J = 7.3 Hz, H-30); 13C NMR(CDCl3): d
14.58, 16.17/16.39, 21.82/21.93, 22.83/22.88, 27.31, 32.08,
34.01/34.16 (d, JCCF = 19 Hz), 35.99/36.08, 36.31/36.40,
43.93, 48.15/48.33 (d, JCCF = 23 Hz), 48.45, 48.47, 71.16,
71.73/72.03 (d, JCCF = 38 Hz), 72.30 (C-22), 91.05/91.12,
99.47/100.87 (d, JCF = 175 Hz), 124.27/124.41, 124.93,
127.21/127.26, 128.16/128.71, 129.62, 133.95/133.96,
136.99/137.04, 147.71, 152.44, 154.05, 171.22, 186.73,
Acknowledgments
The authors thank Dr. Atsuhito Kuboki and Susumu
Ohira (Okayama University of Science) for useful
suggestions.
References and notes
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K. Hayashi et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2470–2474
204.40/204.46; HRFABMS m/z 608.2247 (calcd for
C31H36FNO9Na, 608.2272).
liquid GM containing 20 lM caged ERs (1 or 2) or
50 lM of caged DEX (3) for 30 min. After washing with
a fresh GM, the seedlings were placed on GM agar
solidified over slide glass. The UV light was irradiated to
root on a spot for 3 s by a fluorescent microscope
(Olympus BX50) with a UV-band pass filter (360–
370 nm). The irradiated seedlings were transferred to a
GM agar plates and then incubated vertically for an
additional 12 h in darkness. For control treatment, 5-
day-old transgenic or wild-type seedlings were transferred
to GM agar plate containing 0, 1 lM ER or 10 lM DEX
and grown vertically for 12 h. The GFP expression was
monitored by a fluorescent microscope (Olympus SZX12
or Olympus BX50: Ex 460–490 nm, Em > 510 nm) and
recorded with a digital camera.
15. Photo-conversion of caged steroid hormones to steroid
hormones. Fifty micromolar solution (50% aqueous
MeOH) of caged steroid hormones was irradiated by a
6 W UV lamp equipped with a band pass filter (350–
400 nm) at a distance of 10 cm from solution. The
irradiated solution was analyzed by HPLC at regular
intervals (tR = 8.84 min for ER, 16.49 min for 1: ODS-80T
200 mm · 4.6 mm, MeOH/H2O 9:1, flow rate 0.5 ml/min.
UV detection at 210 nm. tR = 14.31 min for DEX,
43.93 min for 3: ODS-80T, MeOH/H2O 75:25, flow rate
0.5 ml/min. UV detection at 210 nm). The quantum yield
of caged compounds was determined by a direct compar-
ison with that of caged benzonate, 2-nitrophenylethyl
benzoate, in same photolysis condition. 2-Nitrophenyleth-
yl benzoate was synthesized and used as a standard. The
photochemical properties and quantum yield of 2-nitro-
phenylethyl benzoate have been well characterized by Zhu
et al.24 Caged compound solution (50% aqueous MeOH)
was irradiated by UV light (365 nm) with a fluoropho-
tometer (Shimadzu RF-1500). The residual amount of
caged compound in the photolyzed solution was analyzed
by HPLC. The molecular extinction coefficient of 1 and 3
at 365 nm was determined to be 182 and 188 (Mꢀ1 cmꢀ1),
respectively.
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16. Light-regulation of gene expression and development in
transgenic plants. Arabidopsis transgenic seedlings with
pER8::GFP or pIAA14::GFP-mIAA14-GR were grown
for 5 days on vertically oriented germination medium
agar plates (GM, 0.5· Murashige and Skoog salts
[Gibco-BRL, Gaithersburg, MD], 1% sucrose, 1· B5
vitamins, and 0.2 g/L 2-(4-morpholino)-ethane sulfonic
acid (Mes), pH 5.8). The seedlings were immersed in a