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(Fig. 2A). The photolyzed caged dihydrozeatins (1a and 1b) in-
duced ARR5::GUS expression (Fig. 2A). However, the expression
was activated without the photolysis of caged cytokinins (1a and
1b), even though both compounds were designed to demonstrate
esterase resistance. This result was confirmed by the quantitative
measurement of the fluorescent ARR5::GUS reporter activity
(Fig. 2B). These results suggested that the carbonate linkages in
the caged cytokinins (1a and 1b) would have been hydrolyzed in
planta to release dihydrozeatin (7) without photolysis. It is unlikely
that the caged dihydrozeatins (1a and 1b) were active forms of
cytokinin because the introduction of the bulky groups into the
side chain should have abolished the binding affinity to the recep-
tors (Fig. 1B).
An alternative caging approach involved the modification of the
amino group in the adenine moiety of cytokinin (Scheme 1). 2-
Nitrobenzaldehyde was reduced with sodium borohydride and then
brominated with triphenylphosphine and tetrabromomethane in
THF to yield 2-nitrobenzyl bromide. The 2-nitrobenzyl bromide
was reacted with 6-chloropurine and sodium hydride in THF at
room temperature for 12 h to yield caged chloropurines (7a and
7b). These intermediates (7a and 7b) were refluxed with benzyl-
amine or 4-amino-2-methyl-butan-1-ol in butanol for 12 h to yield
N-caged benzyladenine (2a and 2b) and N-caged dihydrozeatin (3a
and 3b). To examine the cytokinin activity and uncaging efficiency
of the caged cytokinins (2a–3b), the cytokinin-responsive ARR5::-
GUS line was used for the assay. The caged dihydrozeatin and ben-
zyladenine (2a–3b) did not induce ARR5::GUS expression without
photolysis (Fig. 2A), suggesting that the N-caged cytokinins (2a–
3b) were inactive analogs and stable in planta. Upon UV irradiation,
the caged cytokinins were uncaged to release the active cytokinins,
benzyladenine (6) and dihydrozeatin (7), thereby activating the
ARR5::GUS expression (Fig. 2A and B).
We further investigated the 4,5-dimethoxy-2-nitrobenzyl
(DMNB) caged cytokinins (2b and 3b). The DMNB moiety exhibited
a higher molecular absorption coefficient at long-wavelength UV
(at 360 nm, the wavelength for uncaging) than the 2-nitrobenzyl
moiety (2a and 3a) due to the bathochromic effects of dimethoxy
groups, indicating that the DMNB caged group could be uncaged
more efficiently than the 2-nitrobenzyl group. To confirm the re-
lease from the caged cytokinins (2b and 3b) by UV irradiation
in vitro, an aqueous methanol solution of caged cytokinin was
photolyzed with a fluorometer at 360 nm (10 nm band-pass), and
the uncaged cytokinin solution was analyzed by HPLC at regular
intervals. Figure 3 shows that the UV irradiation uncaged the cyto-
kinins (2b and 3b) in a time-dependent manner. After 90 min of
exposure, the uncaging yields reached maxima of 27% (2b) and
28% (3b). These results indicated that the cellular cytokinin levels
could be optically controlled by the uncaging rate of the caged
cytokinins.
Figure 4. Manipulation of intracellular cytokinin levels. (A) Photo-controlled
cytokinin-responsive gene expression using caged cytokinin system. Arabidopsis
ARR5::GUS plant was incubated in media containing 20 lM caged cytokinins (2b)
and (3b) for 30 min, and the seedlings were then washed with fresh media to
remove any caged cytokinins outside the cells. The seedlings were placed in agar
plates, and the intracellular caged cytokinins were uncaged using UV light. The
induced GUS enzyme was visualized by histochemical GUS staining. (B) Photo-
controlled root growth using caged cytokinin system. Four-day-old seedlings were
irradiated with UV light in agar media after loading them with caged cytokinin or
benzyladenine (6). The seedlings were vertically cultured at 24 °C in the dark after
photolysis. Photographs of root were taken at 0 h (left panel) and 20 h incubation
(right panel).
We next demonstrated the photo-controlled manipulation of
intracellular cytokinin levels (Fig. 4A). Arabidopsis ARR5::GUS roots
were photo-irradiated after the incorporation of caged cytokinins
into the cells. The seedlings were incubated in liquid GM media
We next examined the photo-control of the physiological plant
cytokinin response (Fig. 4B). Cytokinin inhibits primary root
growth, which is well known to be a rapid physiological response
to exogenous cytokinin.23 Therefore, we studied the root inhibitory
response to investigate the photo-regulation of the cytokinin re-
sponse (Fig. 4B). The Arabidopsis seedlings were placed vertically
in agar media after loading caged benzyladenine (2b) into the
roots. Next, UV light was irradiated to the root and then the seed-
ling was vertically incubated in the dark for 20 h. Benzyladenine
(6) inhibited the root growth, and caged benzyladenine (2b) also
inhibited the root growth to the same extent after photolysis
(Fig. 4B). The roots that were not photo-irradiated showed no ef-
fects with regard to root growth. These results demonstrated that
the physiological cytokinin response of plant can be controlled
by light using the caged cytokinin system.
containing caged cytokinins (20 lM) for 30 min to load the caged
cytokinin, and the seedlings were then washed with fresh GM
media to remove any caged cytokinins outside the cells. The seed-
lings were placed in GM agar media on a glass slide, and the roots
were immediately irradiated with UV light for a few seconds with a
fluorescence microscope. The seedlings on the glass slide were
then incubated in the dark for 7 h to induce ARR5::GUS expression.
Both caged cytokinins (2b) and (3b) were uncaged intracellularly
to activate the ARR5::GUS expression after the incorporation into
the plant cells, but no ARR5::GUS expression was observed without
photolysis (Fig. 4A). Therefore, these results clearly demonstrated
that the caged cytokinin system could manipulate intracellular
cytokinin levels.
The transport of cytokinin involves a critical regulatory system
during plant development; however, the physiological role of
Hayashi, Ken-Ichiro
