Development of 4-methoxy-7-nitroindolinyl (MNI)-caged auxins which are extremely stable in planta

Article abstract of DOI:10.1016/j.bmcl.2015.09.001

Phytohormone auxin is a master regulator in plant growth and development. Regulation of cellular auxin level plays a central role in plant development. Auxin polar transport system modulates an auxin gradient that determines plant developmental process in

Full text of DOI:10.1016/j.bmcl.2015.09.001

Bioorganic & Medicinal Chemistry Letters 25 (2015) 4464–4471
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of 4-methoxy-7-nitroindolinyl (MNI)-caged auxins
which are extremely stable in planta
a,
⇑
a
a
b
a
Ken-ichiro Hayashi , Naoyuki Kusaka , Soma Yamasaki , Yunde Zhao , Hiroshi Nozaki
a
Department of Biochemistry, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA 92093-0116, United States
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 June 2015
Revised 9 July 2015
Accepted 1 September 2015
Available online 1 September 2015
Phytohormone auxin is a master regulator in plant growth and development. Regulation of cellular auxin
level plays a central role in plant development. Auxin polar transport system modulates an auxin gradient
that determines plant developmental process in response to environmental conditions and developmen-
tal programs. Photolabile caged auxins allow optical control of artificial auxin gradients at cellular
resolution. Especially, two-photon uncaging system achieves high spatiotemporal control of photolysis
reaction at two-photon cross-section. However, the development of caged versions of auxin has been
limited by the instability of the caged auxins to higher plant metabolic activities. Here, we describe
the synthesis and application of highly stable caged auxins, 4-methoxy-7-nitroindolinyl (MNI)-caged
auxins. Natural auxin, indole 3-acetic acid, and two synthetic auxins, 1-NAA and 2,4-D were caged by
MNI caging group. MNI-caged auxins showed a high stability in planta and a rapid release the original
auxin when photolyzed. We demonstrated that optical control of auxin-responsive gene expression
and auxin-related physiological responses by using MNI-caged auxins. We anticipate that MNI-caged
auxins will be an effective tool for high-resolution control of endogenous auxin level.
Keywords:
Plant hormone
Auxin
Caged auxin
Two-photon uncaging
Ó 2015 Elsevier Ltd. All rights reserved.
The plant hormone auxin plays a crucial role in almost every
aspects of plant development including embryogenesis, cell elon-
gation, vascular tissue differentiation, tropic responses to light
and gravity, and lateral branching of shoots and roots.1,2 The cellu-
lar auxin levels are modulated by auxin biosynthesis, auxin polar
transport, and auxin catabolism. Indole-3-acetic acid (IAA), the
major naturally occurring auxin, is biosynthesized from trypto-
phan in the indole 3-pyruvate pathway and is polar transported
carrier proteins, and ATP-binding cassette group B (ABCB) auxin
transporters, coordinately modulate auxin transport. The cellular
4
level and localization of auxin transport proteins on plasma mem-
brane was thought to determine the direction and rate of auxin
movement to generate asymmetric auxin gradient in plant tissue.
Furthermore recent studies demonstrated that locally synthesized
auxin also participates in the formation of auxin gradients, and cel-
lular auxin levels are preciously maintained by auxin inactivating
enzymes, such as GH3, IAA-amino acid conjugating enzymes and
1
–3
to generate auxin concentration gradients in plant tissues.
5
–8
Auxin polar streams function in the establishment of the embry-
onic development, lateral development, and vascular patterning.
Additionally, asymmetric redistribution of auxin in response to
environmental and developmental cues drives the asymmetric
tropic growth that allows plants to maintain positioning against
the gravity vector and light. Therefore, spatiotemporal information
of auxin levels would be crucial for dissecting the underlying
auxin oxidases.
These studies indicated that auxin gradients
would not only be formed by transport machinery, but also modu-
lated by complicated processes involving local biosynthesis and
inactivation of auxin. Visualization of cellular auxin gradients
using auxin-responsive reporter lines has been widely utilized to
dissect the role of auxin gradient formation in physiological
9
–11
response of plant.
As an alternative approach, using an artificial
3,4
mechanisms of the physiological roles of auxin gradients.
auxin gradient system would benefit studies of the positional
impact of auxin levels on diverse type of cells and tissue in plants.
A caged compound is an inactivated bioactive molecule and
can rapidly release the original active molecule upon photolysis
Molecular biology and genetic studies have unraveled that the
AUX1 family of auxin influx symporters, PIN family of auxin efflux
of a photo-cleavable protecting group (caging group) with opti-
⇑
Corresponding author. Tel.: +81 86 256 9661; fax: +81 86 256 9559.
E-mail address: hayashi@dbc.ous.ac.jp (K.-i. Hayashi).
12,13
cal devices.
With caged compounds, the uncaging reaction
can be spatiotemporally controlled at cellular level by precious
control of light-irradiation. Additionally, the active molecule is
URL:
K.-i. Hayashi).
http://www.dbc.ous.ac.jp/labs/KHayashi/KhayashiEnglish.html
(
http://dx.doi.org/10.1016/j.bmcl.2015.09.001
960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
0

K.-i. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4464–4471
4465
intracellularly released, which can be repeated at any point during
an experiment without physical perturbing. The intracellular levels
of the released active molecule can be precisely controlled by the
near infrared light (760 nm) used in two-photon excitation can
reach into deeper tissue compared with UV light in one-photon
excitation (360 nm). However, higher concentrations of caged
molecules are required due to the low uncaging efficiency by
two-photon excitation. We herein demonstrate the high stability
of MNI-caged auxins in Arabidopsis plants and the photo-triggered
spatiotemporal controls of auxin response in roots by one-photon
excitation system.
1
2,14
0
0
rate of uncaging.
We previously demonstrated that 2 ,5 -
dimethoxyphenyl-2-nitrobenzyl (DMPNB)-caged auxins were use-
ful tools to establish an artificial auxin gradient.15 DMPNB-caged
auxin displayed useful features and advantages in comparison with
classical methods of auxin application, such as a solution, a waxy
paste, or an agar block containing auxin. Plant cells generally show
higher metabolic capacity than mammal cells. Therefore, conven-
tional caging groups used for mammalian biology are readily
hydrolyzed by plant enzymes to release auxins without photoly-
Synthesis of MNI-caged auxins: Napthalene-1-acetic acid (NAA)
and 2,4-dichlorophenoxy acetic acid (2,4-D), two well character-
ized synthetic auxins, and the natural auxin IAA are (Scheme 1)
widely used for physiological experiments. NAA and 2,4-D exhib-
ited a different transport profile from IAA. The transport profiles
of the two typical synthetic auxins have been extensively charac-
1
5
sis. DMPNB-caged auxin was designed to be resistant against
esterases and to have considerable stability both in vitro and
in vivo. DMPNB-caged auxins enable the manipulation of cellular
auxin levels within a single cell by fine control of optical devices,
suggesting that an artificial auxin gradient can be achieved at a
desired position by means of a caged auxin system.
3
,4
terized. PIN carrier proteins export NAA to outside of the cell,
but NAA is not actively imported into the cell. In contrast, 2,4-D
4
is imported by AUX1, but is not actively exported. Therefore, these
two synthetic auxins have been used as diagnostic tools for influx
or efflux auxin transport machinery.
In this study, we synthesized MNI (4-methoxy-7-nitroin-
dolinyl)-caged IAA, NAA and 2,4-D in which the carboxylic acid
was protected by a secondary amine of MNI caging group.
MNI-caged auxins showed higher in vivo stability than
DMPNB-caged auxins. Additionally, MNI-caged auxins can be used
for two-photon uncaging system, as the MNI-caging group was
As the initial step of auxin response, auxin is perceived by
TIR1-Aux/IAA receptor complex in Arabidopsis. The crystal struc-
tures of the auxin-bound TIR1-Aux/IAA receptor complex revealed
that aromatic ring and carboxylic acid in auxin molecule are essen-
tial for the affinity to the binding site of the receptor complex.18
Based on the structural information, caging of the carboxylic acid
in auxin would diminish the binding activity of auxin molecules
to yield physiologically inert caged auxin molecules.
Plant cells seem to show higher esterase activities and the ster-
ically accessible ester linkage in caged compound, such as NPE-IAA
(4) are readily hydrolyzed in planta. In our previous studies,
DMPNB-caged auxin was designed to be esterase-resistant caged
originally developed to be a photolabile group for the two-photon
excitation system,1 and the MNI-caged
6
L-glutamate and
MNI-caged-D-aspartate are widely used as commercial reagent
13
for two-photon uncaging in neuroscience. Two-photon uncaging
takes advantage of the high spatial resolution in comparison with
one-photon uncaging, because photolysis was occurred at the
cross-section of the two-photon excitation (Fig. 1A).17 Additionally,
Figure 1. In vitro uncaging of MNI-auxins. (A) A schematic diagram for one-photon and two-photon excitation. (B–D) In vitro uncaging rates of MNI-caged auxins. (B) MNI-
IAA, (C) MNI-NAA (D) MNI-NAA, 100 M MNI-caged auxin solution (85% aqueous EtOH) was irradiated by a fluorophotometer (365 nm), and photolyzed solution were
analyzed by HPLC at regular intervals. The amount of released auxins was plotted as the relative uncaged yield (%) from the initial amount of caged auxins.
l

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K.-i. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4464–4471
Scheme 1. Reagents and conditions: (a) NaBH
and 43% for 10); (d) TFA in CH Cl
acetic acid, SOCl , in toluene, 70 °C, 6 h (81%); (g) TFA in CH
3
3 3
CN in AcOH, rt, 3 h (98%); (b) di-tert-butyl dicarbonate, triethylamine, rt, 3 h (93%); (c) AgNO , AcCl in CH CN, rt, 2 h (41% for 9
2
2
, 0 °C, 0.5 h then rt, 3 h (93%); (e) 1-NAA or 2,4-D, SOCl
2
, in toluene, 75 °C, 8 h (56% for 2 and 63% for 3); (f) N-tert-butoxycarbonyl-indole 3-
2
2
Cl , 0 °C, 0.5 h then rt, 3 h (94%).
2
auxin.15 The introduction of the bulky 1,4-dimethoxyphenyl (DMP)
ring into the 2-nitrobenzyl caging group, effectively blocked the
hydrolysis of the ester of caged auxin by the plant esterases and
consequently enhanced the stability of the caged auxin in vivo.
However, the DMPNB-caged auxins were still slightly hydrolyzed
in planta to release auxins during long-term cultivation. Therefore,
we applied MNI caging group to the caged auxin system. In
MNI-caged auxin, the carboxylic acid in auxin is caged by ternary
amide with MNI group. Thus, MNI-caged auxins are expected to
be completely inert to esterases and are resistant to general
peptidases. Furthermore, in principle, MNI-caged auxin can be
applicable to two-photon uncaging system as MNI caging group
has been originally developed for two-photon excitation.17
MNI-caged auxins are uncaged in vitro by light-irradiation: To
estimate the uncaging efficiency of MNI-caged auxins, 100
l
M
MNI-caged auxins in the aqueous ethanol solution (85% EtOH)
was light-irradiated by fluorometer (365 nm) and the photolyzed
solution at regular intervals was analyzed by HPLC (Fig. 1B).
MNI-IAA was photolyzed to release original IAA molecule in
proportion to irradiation time until 5 min and MNI-IAA was com-
pletely consumed after 30 min irradiation. The release yield of
IAA from MNI-IAA was ca. 30% at 30 min exposure. MNI-NAA and
MNI-2,4-D was also rapidly photolyzed and released original aux-
ins (Fig. 1C and D). Both MNI-NAA and MNI-2,4-D were completely
disappeared by 15-min irradiation. The release yield of NAA and
2,4-D was reached to ca. 60% and 20% at 30-min exposure in our
experimental condition, respectively.
The synthetic scheme of MNI-auxins is shown in Scheme 1.
4
-Methoxyindole (6) was reduced by cyanoborohydride in acetic
MNI-caged auxins are highly stable in plant cells: Next, we vali-
dated the uncaging efficiency of MNI-caged auxin in culture med-
ium and evaluated in vivo stability. The transgenic Arabidopsis
auxin-responsive DR5::GUS reporter line was used for the evalua-
tion of auxin level that was released from the MNI-caged auxins.
The DR5::GUS reporter line contains the b-glucuronidase (GUS)
reporter gene under the control of the synthetic auxin-responsive
promoter DR5, and the expression of GUS reporter enzyme was
specifically controlled by auxin in a dose-dependent manner.20
The auxin-induced GUS enzyme activity was estimated by the
histochemical staining using chromogenic substrate X-Gluc and
was fluorometrically quantified using 4-methyl umbelliferyl-b-
acid to give 4-methoxyindoline (7). This indoline was protected
with tert-butoxycarbonyl (Boc) group by the treatment with di-
tert-butyl dicarbonate and triethylamine in THF.19 The Boc-indo-
line (8) was nitrated with AgNO
yield a mixture of the 5- and 7-mononitro regioisomers (9 and
0), (in 1:1 ratio). 1-N-Boc-7-nitoro-4-methoxyindoline (10) was
then deprotected with trifluoroacetic acid in CH Cl to afford
-nitoro-4-methoxyindoline (11). IAA was reacted with thionyl
3 3
and acetyl chloride in CH CN to
1
2
2
7
chloride and 7-nitoro-4-methoxyindoline (11) in toluene at 70 °C
for 6 h to yield amido (12). This 1-N-Boc-MNI-IAA (12) was depro-
tected with trifluoroacetic acid to give a MNI-caged IAA (1) as pale
yellow oil. Two typical synthetic auxins, NAA and 2,4-D were also
caged by photolabile MNI group. MNI-caged NAA (2) and
MNI-caged 2,4-D (3) were synthesized by the condensation of
D
-glucuronide as a fluorogenic substrate. For the measurement of
the uncaging efficiency in the culture medium, MNI-caged auxins
(20 M) in GM liquid medium was irradiated by using a UV lamp
l
7
-nitoro-4-methoxyindoline (11) and synthetic auxins in the
(360 nm), and the 5-days old DR5::GUS seedling was cultured in
presence of thionyl chloride as shown in Scheme 1.
the photolyzed medium for 5 h in the dark (Fig. 2A–C, light

K.-i. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4464–4471
4467
Figure 2. MNI-caged auxins are highly stable in planta and are uncaged by light-irradiation. (A–C) The caged auxins (20
lM) in the GM culture media were photolyzed by
light irradiation (350–400 nm). 5-days-old Arabidopsis auxin-responsive DR5::GUS seedlings were incubated in the photolyzed medium (+light) or non-irradiated medium
ꢀlight) for 5 h in the dark. The GUS reporter enzyme was induced to the released auxins, and quantified by a fluorometer with a fluorescent substrate. The GUS activity was
shown as normalized values to 100% values of 10 M auxin treatment. Bar represents SD (n = 15). (D) DR5::GUS seedling was incubated with 20 M caged auxin for 24 h in the
dark without photolysis and then auxin-inducible DR5::GUS activity visualized by histochemical b- -glucuronidase (GUS) staining.
(
l
l
D
irradiated). For the evaluation for intracellular stability of caged
auxin, the DR5::GUS seedlings were incubated for 5 h (Fig. 2A–C,
without light) and 24 h (Fig. 2D) in GM medium containing caged
auxins in the dark. In this stability assay, caged auxin molecule
would be constantly diffused from medium into the plant cells.
The intracellular caged auxin would be converted to original auxin

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K.-i. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4464–4471
Figure 3. Uncaging of intracellular caged auxin in planta. 5-days-old DR5::GUS seedling was incubated in caged auxin solution for 30 min and then the seedlings were washed
with fresh medium. After loading of caged auxins, the seedlings were placed on agar medium plate. The intracellular caged auxins were uncaged by light irradiation and then
the seedling was incubated for 5 h in the dark. The auxin-induced GUS enzyme activity was visualized by histochemical blue staining. The values in parenthesis represent the
concentration (lM) of caged auxin solution used for loading.
molecule, if caged auxin is acceptable substrate for the metabolic
conversion catalyzed in planta.
In previous Letter, esterase resistant DMPNB-caged auxins were
demonstrated to be more stable in plant cell than
were hydrolyzed to induce DR5::GUS expression visualized by blue
staining with X-gluc substrate (Fig. 2D). In the control root, DR5::
GUS reporter gene has been expressed at root tip where
endogenous IAA is accumulated (Figs. 2D and 3, mock). Similarly,
MNI-caged auxin did not induce any DR5::GUS expression on root
after 24 h incubation (Fig. 2D). Our results suggest that MNI-auxins
were highly stable in planta and were resistant to the enzymatic
hydrolysis by plant amidases.
We next examined the manipulation of intracellular auxin
levels by using MNI-auxins (Fig. 3). After the incorporation of
MNI-caged auxins into the cells, intracellular MNI-caged auxins
would be uncaged to directly release IAA inside the cells. To load
the MNI-caged auxins to the cell, the 5-days-old DR5::GUS
2
-nitrophenylethyl (NPE)-caged auxin. NPE group is widely used
as conventional caging group in mammal biology, but NPE-IAA
and NPE-2,4-D were unstable in plants. Consistent with previous
Letters, DMPNB-caged auxins released original auxins upon pho-
tolysis, thereby induced DR5::GUS expression (Fig. 2A–C). Similarly,
MNI-auxins in culture medium were uncaged by light to release
original auxins and then lead to activation of DR5::GUS expression.
However, uncaging efficiency of MNI-caged auxins was lower than
those of DMPNB-caged auxins. Without photolysis, NPE-caged
auxins activated DR5::GUS reporter expression after 5 h incubation
seedlings were incubated in 50 and 100 lM MNI-caged auxin
(
(
Fig. 2A–C). Contrary, DMPNB-caged auxin and MNI-caged auxins
20 M) did not activate DR5::GUS expression by 5-h incubation
solution (liquid medium) for 30 min, and then MNI-caged auxins
outside the cells were washed out with fresh medium. The
seedlings were placed on the glass slide covered with agar plate
medium, and the roots were irradiated with a fluorescence micro-
scope equipped with high pressure mercury lamp (>UV350 nm
l
without photolysis, suggesting that both DMPNB- and MNI-caged
auxins were stable in the cell. However, after 24 h incubation
without photolysis, DMPNB-caged IAA and DMPNB-caged 2,4-D

K.-i. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4464–4471
4469
spatiotemporal manipulation of intercellular auxin level in planta,
we used transgenic Arabidopsis DII-VENUS line expressing a trans-
lational fusion protein of the Aux/IAA auxin-interaction domain
(
DII) and yellow fluorescent protein variant (VENUS) expressed
1
0
under a constitutive 35S promoter. Auxin binds to TIR1/AFB
auxin receptors and promotes the interaction between DII-VENUS
TIR1
and SCF
E3 ligase complex. DII-VENUS fusion protein is ubiqui-
tinated in an auxin-dependent manner and rapidly decomposed
via the ubiquitin–proteasome pathway. The amount of DII-VENUS
protein in cell indirectly reflects the intracellular auxin concentra-
1
0
tion. The DII-VENUS seedlings were treated with auxin biosyn-
2
1
22
thesis inhibitors, L-kynurenine (5 lM) and yucasin (50 lM)
for 5 h. Because native DII-VENUS fluorescent image in root is
already a reflect of endogenous IAA distribution. The DII-VENUS
protein was uniformly accumulated and distributed after the inhi-
bition of endogenous IAA production by the inhibitors. The DII-
VENUS seedlings were then incubated with MNI-IAA for 20 min
and then the root was immediately irradiated by light (Fig. 4).
The time series displayed that the VENUS fluorescence in the
nucleus disappeared by whole root irradiation of MNI-IAA treated
root (Fig. 4B) and IAA treatment (Fig. 4C), whereas the degradation
of DII-VENUS fluorescent signals in MNI-IAA treated root was lim-
ited within at light-irradiated area (Fig. 4A). Thus, MNI-caged auxin
system can spatiotemporally manipulate intracellular auxin levels
in planta by controlling light illumination.
We next investigated the light-control of auxin-regulated phys-
iological responses (Fig. 5). Auxin represses primary root growth
that is a typical rapid response to exogenous auxin. The Arabidopsis
seedlings were vertically placed on agar medium after loading
MNI-IAA into the roots. The seedling was vertically incubated in
the dark for 5 h after light irradiation (5 min, UV350–360 nm).
UV light alone did not affect the primary root growth (Fig. 5A).
MNI-IAA inhibited the root growth after light irradiation (Fig. 5A),
but did not without the irradiation.
IAA is biosynthesized from tryptophan by two enzymes, the
tryptophan aminotransferase TAA1 and YUCCA (YUC) enzymes in
the Indole-3-pyruvate (IPA) pathway. YUCCA, flavin-monooxyge-
nases catalyze the conversion of IPA to IAA and function as a
rate-limiting enzyme in IPA pathway. Arabidopsis has 11 YUC
family genes and the disruption of root expressing 5 YUC genes
(
yucca 3 5 7 8 9) showed severe auxin deficient root phenotypes.6
Especially, yucca quintuple (yuc Q) mutants show clear defects in
6
root gravitropism and root hair formation. MNI-IAA was loaded
to wild-type and yuc Q mutant plants and then intracellular
MNI-IAA was uncaged on the GM agar plate by light irradiation.
MNI-IAA promoted the root hair formation in wild-type root by
photolysis. Furthermore, the defects in root hair formation and
root gravitropic response (winding root) in yuc Q mutants were
recovered by uncaging of MNI-IAA, suggesting that the MNI-IAA
can manipulate endogenous auxin levels without toxic effects of
uncaged MNI group on root development. This result demon-
strated that the physiological auxin responses can be controlled
by a light using the MNI-caged auxin system.
Figure 4. Spatiotemporal manipulation of cellular auxin level in planta. 5-days-old
DII-VENUS root was incubated in 10 M MNI-IAA solution for 20 min. The roots
were immediately irradiated by light (360 nm) as a spot (A) or whole root (B). The
roots were immersed in 2 M IAA (C). The degradation of nuclear-localized DII-
VENUS protein was monitored at 2, 20, and 40 min after exposure and IAA
treatment. Bar represents 100 m.
l
l
l
pass filter) for a few seconds. The DR5::GUS seedlings on the glass
slide were then cultured for additional 5 h in the dark.
MNI-caged auxins were uncaged intracellularly to release
auxins and activated the DR5::GUS expression, but no GUS reporter
Caged auxin system is a promising approach to establish an
artificial auxin gradient in a defined spatiotemporal manner. To
investigate a physiological function of a spatiotemporal regulation
of auxin gradient modulated by polar auxin transport, it is crucial
to manipulate the cellular distribution of auxin. Two-photon unca-
ging system takes significant advantages of the high spatial resolu-
expression was observed without photolysis at 100
condition (Fig. 3). Contrary, NPE-caged auxins and DMPNB-caged
auxins released auxin by light irradiation. However, over 50
lM loading
lM
1
3,17
loading condition, esterase-resistant DMPNB-caged auxins were
slightly hydrolyzed without photolysis. This result clearly demon-
strated that the MNI-caged auxin can be highly stable in the plant
cell and could modulate the endogenous auxin level by controlling
light irradiation.
tion.
However, cellular level of caged molecule in two-photon
system requires considerably higher concentration than one-pho-
ton system. Therefore, caged molecule in two-photon system must
be inert to metabolic pathways in cell and be non-toxic to the cell.
MNI-caged auxins would satisfy these requirements to perform the
two-photon uncaging by two-photon fluorescent microscopy. Very
recently, DMPNB-caged auxin derivative in combination with
MNI-caged auxins alter the auxin-regulated physiological
responses in Arabidopsis plants: To examine consequences of

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K.-i. Hayashi et al. / Bioorg. Med. Chem. Lett. 25 (2015) 4464–4471
Figure 5. Manipulation of physiological auxin response by light irradiation using MNI-IAA. (A) 5-days-old seedlings were incubated in 10
After washing with fresh medium, seedlings were irradiated on the agar medium plate for 5 min with light (350–400 nm). After photolysis, the seedlings were cultured
vertically in the dark. Photograph was recorded at regular intervals. (B) 6-days-old yuc Q (yuc 3 5 7 8 9) mutant and wild type seedling were incubated in 50 M MNI-IAA
solution for 20 min and then uncaged with light. The seedlings were vertically cultured in the dark after photolysis for 15 h. Bar represent 10 mm and 1 mm each.
lM MNI-IAA solution for 20 min.
l
auxin-inducible degradation system demonstrated light-controlled
spatiotemporal regulation of conditional proteolysis in mammal
cell. MNI-caged auxins might be directly applicable to these
mammal system. We anticipate that MNI-caged auxins would
be useful tools for spatiotemporal regulation for auxin gradient
leading to the control of plant development.
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