Nanometer-scale direct observation of the receptor for the leaf-movement factor in plant cell by a novel TEM probe

Article abstract of DOI:10.1016/j.tetlet.2006.12.128

Electron microscopy is a useful method for observing localization of some proteins in a cell. In this Letter, we report a nanometer-scale direct observation of the receptor for a bioactive substance of small molecular weight by using TEM (transmission ele

Full text of DOI:10.1016/j.tetlet.2006.12.128

Tetrahedron Letters 48 (2007) 1341–1344
Nanometer-scale direct observation of the receptor for the
leaf-movement factor in plant cell by a novel TEM probe
Yoshiyuki Manabe,a Takanori Sugimoto,a Tomoyuki Kawasakib and Minoru Uedaa,*
aDepartment of Chemistry, Faculty of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
bFaculty of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
Received 17 November 2006; revised 18 December 2006; accepted 21 December 2006
Available online 23 December 2006
Abstract—Electron microscopy is a useful method for observing localization of some proteins in a cell. In this Letter, we report a
nanometer-scale direct observation of the receptor for a bioactive substance of small molecular weight by using TEM (transmission
electron microscopy). We developed a novel TEM probe compound (1) that is a modified leaf-movement factor with benzophenone
as a photoaffinity group and FITC as an antigen that is recognized by a nano-gold bound anti-FITC antibody. By using probe 1, we
revealed that the receptor for the leaf-movement factor of Cassia mimosoides is localized in the plasma membrane of the plant motor
cell.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Most leguminous plants close their leaves in the evening,
as if to sleep, and open them in the morning according
to the circadian rhythm controlled by a biological clock.
Charles Darwin, well-known for his theory of evolution,
carried out the pioneering work in this field.1 And in the
1970s, physiological studies revealed that nyctinastic leaf
movement is induced by the swelling and shrinking of
motor cells in the pulvini, a small organ located in the
joint of the leaf to the stem.2 Flux of potassium ions
across the plasma membranes of the motor cells is fol-
lowed by massive water flux, which results in swelling
and shrinking of these cells. We revealed that nyctinasty
is controlled by a pair of leaf-movement factors: leaf-
opening and leaf-closing substances.3,4 Recently, we
have revealed that a receptor for the leaf-movement fac-
tor is involved in the membrane fraction of the motor
cell by using a photoaffinity-labeling method.5 Photo-
affinity experiment using a crude membrane fraction of
Cassia plant gave two potential receptors of 210 and
180 kDa.5 However, the crude membrane fraction that
was used in the experiment contained both a vacuolar
membrane and a plasma membrane. It was extremely
difficult to separate them in such a small scale. Thus,
it was impossible to determine which membrane
contains the receptor for the leaf-movement factor.
Determination of the localization of the receptor is very
important in the bioorganic study of nyctinasty (Fig. 1):
If the receptor is involved in the plasma membrane, the
leaf-movement factor is an inter-cellular chemical
messenger and controls the activity of the potassium
channel in a plasma membrane (Fig. 1a). On the other
hand, if the receptor is involved in the vacuolar
Keywords: Nyctinasty; Leaf-opening substance; TEM; Nano-gold;
TEM probe.
*
Figure 1. Two possible mechanisms in the turgor control of the motor
cell by potassium isolespedezate (2).
Corresponding author. Tel./fax: +81 22 795 6553; e-mail: ueda@
mail.tains.tohoku.ac.jp
0040-4039/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.12.128

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Y. Manabe et al. / Tetrahedron Letters 48 (2007) 1341–1344
membrane, the leaf-movement factor is an intra-cellular
chemical messenger that activates the potassium channel
in the vacuolar membrane which triggered the flux of
potassium ions across plasma membranes (Fig. 1b). To
examine these two possibilities, we tried to identify the
direct observation of the exact localization of the recep-
tor in the motor cell. However, it required the observa-
tion with nanometer-scale resolution. Thus, we thought
that a transmission electron microscopy (TEM) which
has a resolution of 0.10 nm would be suitable for this
purpose.
In this Letter, we report the design and synthesis of a
novel TEM probe compound (1) that enables the nano-
meter-scale direct observation of the localization of the
receptor for 1.
The TEM probe has a photoaffinity labeling group and
antigen for immunoelectronmicroscopy (Fig. 2). After
cross-link formation to the receptor by photoaffinity
labeling, the TEM probe will act as an antigen which
can be recognized by a nano-gold5 bound corresponding
antibody. Then, we can label the receptor for 2 by a
nano-gold which can be monitored by using TEM.
Based on this concept, TEM probe (1) was designed
according to the results of structure–activity relationship
studies on 2.6,7 Structure modification in the sugar moi-
ety of 2, such as conversion into galactoside, a-manno-
side, and even L-glucoside, did not cause any decrease
in bioactivity. These results suggested that there would
be little structure recognition by the receptor on the
sugar moiety of 2. Thus, retention of bioactivity can
be expected after structural modification in the sugar
moiety of 2. Benzophenone was introduced in the 20-po-
Figure 2. Concept of TEM probe: photoaffinity antigen-tagging of
receptor on cell membrane.
Scheme 1. Synthesis of TEM probe (1).

Y. Manabe et al. / Tetrahedron Letters 48 (2007) 1341–1344
1343
sition of the sugar moiety as a photoaffinity group and
FITC was introduced in the 60-position as an antigen.
The combination of benzophenone and FITC is very
important. No photobleaching of the photoaffinity
labeling group which is induced by an intramolecular-
electron transfer was observed among them. It was sup-
posed that other photolabeling groups, such as trifluoro-
methyldiazirine, whose absorption spectrum has an
overlap with the fluorescence spectrum of FITC, cannot
form a cross-link with a receptor efficiently because of
the photobleaching due to the intramolecular electron
transfer from the photoaffinity group to FITC.
We synthesized probe 2 according to the synthesis of the
photoaffinity probe.8 Synthetic intermediate 35 was
reduced and the resulting amine was coupled with
Boc-protected glycylglycylglycine. After deprotection,
stepwise introduction of benzophenone and then FITC
was carried out to give TEM probe (1)9 (Scheme 1).
Figure 4. Preparation of nano-gold bound Fab0 fragment of anti-
FITC antibody.
Inc.) (Fig. 4).5,10 Anti-FITC antibody F(ab0)2 was re-
duced by cysteamine. The resulting Fab0 fragment was
purified by FPLC, and coupled with monomaleimido
nano-gold, and used in labeling of the plant section.
We prepared a section of the plant pulvini which
contains a motor cell in 30 lm-thickness. After being
incubated with 1 · 10ꢀ4 M aqueous solution of TEM
probe (2) at 4 ꢁC for 4 h, the section was irradiated by
UV (365 nm) to form a cross-linkage between probe 1
and its receptor. Then the section was washed by Milli
Q thoroughly to remove the unreacted 1. After washing
thoroughly, the cross-link formation was examined by
the observation of fluorescence on the plant section by
using a fluorescence microscope (Fig. 3). Cross-linked
1 cannot be removed from the plant section by the wash-
ing and gave fluorescence due to the fluorescein moiety
in 1. Without UV irradiation, all of 1 was removed by
washing and no fluorescence was observed on the plant
section. Moreover, the specific binding of 1 was con-
firmed by the competitive binding experiment in the
presence of 1000-fold amount (1 · 10ꢀ1 M) of non-
labeled natural product (2). As shown in Figure 3,
decrease in the strength of fluorescence around the
motor cell was observed under this condition.
The section was then fixed with 4% glutaraldehyde and
0.5% formaldehyde. The HQ Silver Enhancement-Kit
(Nanoprobe Inc.) for silver enhancement was also used
to improve the sensitivity in TEM observation.5 After
dehydration with a series of EtOHaq, the section was
embedded with Spurr resin. Finally, the embedded sec-
tion was cut to a thickness of about 90 nm, stained by
UO2(OAc)2, and used for the observation by TEM (Hit-
achi H-8100).
Figure 5 shows the TEM image of the plasma membrane
of the motor cell. FITC-tagged receptors were labeled
by nano-gold and can be observed as black stain in
the plasma membrane. From this result, the receptor
for 1 is confirmed to be localized in the plasma mem-
brane of the motor cell; thus the proposed mechanism
in Figure 1a is correct: the receptor of 2 is involved in
the plasma membrane, 2 is the inter-cellular chemical
messenger and controls the activity of the potassium
channel in the plasma membrane of the motor cell.
The cross-linked section was treated with the nano-gold
bound Fab0 fragment of the anti-FITC antibody. To
improve the penetration of the antibody into the plant
section of 30-lm thickness, Fab0 fragment was used as
an antibody which was prepared from commercially avail-
able anti-FITC antibody F(ab0)2 (from Goat, Rockland
This concept can be widely applicable for the nanometer
scale-direct observation of the receptor for any small
molecules.
Figure 3. Fluorescence image of plant pulvini containing motor cell. Cross-link formation was examined by fluorescence of 1 on the motor cell.
Strong fluorescence observed in the center of each photograph is due to the non-specific binding of 1 to the plant vessel (left: cross-linked 1, center:
without irradiation, right: under competitive binding with 2).

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Y. Manabe et al. / Tetrahedron Letters 48 (2007) 1341–1344
References and notes
1. Darwin, C. The Power of Movement in Plants. Third
Thousand; John Murray: London, 1882.
2. Satter, R. L.; Gorton, H. L.; Vogelmann, T. C. The
Pulvinus: Motor Organ for Leaf Movement; American
Society of Plant Physiologists, 1990.
3. Ueda, M.; Yamamura, S. Angew. Chem., Int. Ed. 2000, 39,
1400–1414.
4. Ueda, M.; Nakamura, Y. Nat. Prod. Rep. 2006, 23, 548–
557.
5. Hainfeld, J. F.; Powell, R. D. J. Histochem. Cytochem.
2000, 48, 471–480.
6. Shigemori, H.; Sakai, N.; Miyoshi, E.; Shizuri, Y.;
Yamamura, S. Tetrahedron 1990, 46, 383–394.
7. Ueda, M.; Sawai, Y.; Yamamura, S. Tetrahedron Lett.
1999, 40, 3757–3760.
8. Fujii, T.; Manabe, Y.; Sugimoto, T.; Ueda, M. Tetra-
hedron 2005, 61, 7874–7893.
9. Compound 1: 1H NMR (300 MHz, CD3OD, rt): 8.33 (1H,
br s), 7.80–7.61 (10H, m), 7.48 (2H, t, J = 7.7 Hz), 7.22
(1H, s), 7.15 (1H, d, J = 8.3 Hz), 6.76–6.66 (6H, m), 6.57
(2H, m), 5.24 (1H, d, J = 8.4 Hz), 4.73 (1H, d,
J = 12.8 Hz), 4.51 (1H, d, J = 12.8 Hz), 4.30 (2H, s),
4.06 (1H, dd, J = 3.3, 10.8 Hz), 3.88 (3H, s), 3.74 (2H, s),
3.69 (1H, t, J = 7.0 Hz), 3.60 (1H, dd, J = 8.4, 10.8 Hz)
ppm; 13C NMR (75 MHz, CD3OD, rt): 197.7, 184.2,
173.4, 172.4, 170.7, 169.2, 168.0, 162.4, 160.8, 154.8, 142.1,
139.7, 139.1, 138.4, 137.1, 134.5, 134.1, 131.9, 131.5, 131.4,
130.1, 130.8, 130.1, 129.6, 126.5, 124.4, 112.1, 103.5, 101.1,
89.2, 74.6, 70.5, 68.9, 61.6, 52.2, 44.1, 43.3, 40.2, 30.8,
22.9 ppm. HR ESI MS (positive): [M+H]+ found m/z
1095.3083, C56H50N6O16S requires m/z 1095.3082; IR
19
(film) m: 3294, 1654, 1605, 1541, 1512 cmꢀ1; ½aꢁD ꢀ7.1 (c
0.50, MeOH).
10. Anti-FITC F(ab0)2 fragment (0.2 mg in 167 lL, Goat-
Poly, Rockland) was diluted by 5 mM EDTA—0.1 M
phosphate buffer (pH 6.0) to 1 mL, and then cysteamine
hydrochloride (6.0 mg, Aldrich) was added to this solu-
tion. After being allowed to stand at rt for 1 h, the reaction
mixture was separated by using gel filtration using Fast
Desalting Column HR 10/10 (Amersham) with 0.02 M
sodium phosphate (pH 6.5) containing 1 mM EDTA and
0.15 M NaCl, and then concentrated with Centricon YM-
10 (Millipore). Aqueous solution of monomaleimido
nano-gold (250 lL, Nanoprobe) was added to the result-
ing Fab0 fragment (0.1 mg), and then allowed to stand at
4 ꢁC overnight. The solution was concentrated with
Centricon YM-30 (Millipore), and then separated by
Superdex 75 HR 10/30 with 0.02 M sodium phosphate
(pH 7.4) containing 0.15 M NaCl to give nano-gold-bound
anti-FITC Fab0 fragment.
Figure 5. TEM image of plant motor cell (upper: TEM image of plant
motor cell, under: expansion of square part in upper photograph).
Acknowledgments
This work was supported by Grant-in-Aid for Scientific
Research on Priority Area 16073216 from the Ministry
of Education, Science, Sports, and Culture (MEXT),
Grant-in-Aid for Scientific Research (No. 15681013).
We also thank the Experimental Station for Medicinal
Plant Studies, Graduate School of Pharmaceutical
Sciences, Tohoku University, for the cultivation of
Cassia plant. And we thank Professor Masayuki Inoue
(Tohoku University) for useful discussion on the use
of nano-gold particle.