The biological significance of leaf-movement, an approach using a synthetic inhibitor of leaf-closure

Article abstract of DOI:10.1016/S0040-4039(02)01770-7

Nyctinastic leaf-movement, which is folding and opening movement of the leaves according to circadian rhythm, have been widely observed in leguminous plants. Although this movement has been known since the age of Alexander the Great, the question 'Why doe

Full text of DOI:10.1016/S0040-4039(02)01770-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 7545–7548
The biological significance of leaf-movement, an approach using
a synthetic inhibitor of leaf-closure
Minoru Ueda,* Takanori Sugimoto, Yoshiyuki Sawai and Shosuke Yamamura
Laboratory of Natural Products Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1,
Yokohama 223-8522, Japan
Received 1 July 2002; revised 6 August 2002; accepted 23 August 2002
Abstract—Nyctinastic leaf-movement, which is folding and opening movement of the leaves according to circadian rhythm, have
been widely observed in leguminous plants. Although this movement has been known since the age of Alexander the Great, the
question ‘Why does leguminous plant sleep?’ has always puzzled many scientists studying nyctinasty. We have revealed that
nyctinastic leaf-movement is essential for the survival of leguminous plants by inhibiting leaf-movement using synthetic leaf
movement inhibitor. Leguminous plant will wither and die without nyctinasty. Our result gives an important clue for the historic
mystery, ‘Why does the leguminous plant sleep?’ © 2002 Elsevier Science Ltd. All rights reserved.
Plants are unable to move from one place to another.
However, folding and opening movement of the leaves
according to circadian rhythm have been widely
observed in leguminous plants. This periodic leaf-move-
ment is called nyctinasty and has been known since the
age of Alexander the Great.1 On the other hand, the
question ‘Why does the leguminous plant sleep?’ has
always puzzled many scientists studying nyctinasty.
Darwin concluded that nyctinasty provided protection
from chilling in addition to actual freezing,2 whereas
later studies made important objections against his
hypothesis.3,4 Bu¨nning proposed that nyctinasty pro-
tected the photoperiodic time keeping system from
moonlight, because moonlight falling on leaves during
night might prevent accurate measurement of night
length.5 However, no experimental evidence to date has
been reported that explains the biological significance
of nyctinasty. Research has been hindered because we
could not inhibit the leaf movement. Also, no mutant
without nyctinasty has been reported so far. Thus,
genetic approach to this issue will be difficult. Now we
have succeeded in inhibiting leaf movement using syn-
thetic inhibitor of leaf closure based on the substance
that naturally induces leaf opening.6 Our result provide
the first experimental evidence to answer the question
‘Why does the leguminous plant sleep?’
Schildknecht’s turgorin had been widely believed to be
a endogeneous factor controlling leaf movement,1 but
we revealed that it did not show any bioactivity under
physiological conditions.6,7 Nyctinasty is controled by
two endogenous factors of contrasting bioactivities:
leaf-closing substance which makes the leaf closed and
leaf-opening substance which makes the leaf open.6 The
Figure 1. The control of internal concentration of 1 by a biological clock that causes the nyctinastic leaf-movement.
* Corresponding author.
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01770-7

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M. Ueda et al. / Tetrahedron Letters 43 (2002) 7545–7548
bioactivity of these factors is extremely specific to the
original plant from which they were isolated. In the
plant body, the rhythm of nyctinasty is generated by
change in balance of concentrations between two leaf-
movement factors according to a circadian rhythm.6
This change in balance can be attributed to the hydroly-
sis of glucoside-type leaf-movement factor into the cor-
responding aglycon by b-glucosidase, whose activity is
controlled by a biological clock (Fig. 1).6
stem of C. mimosoides and placed in H2O kept doing
the circadian rhythmic leaf movement (Fig. 2). Both of
leaf-opening substance (1) and leaf-movement
inhibitors (2, 4, and 5) can keep the leaves open even at
night at 1×10−6 mol/l. When the leaves were treated
with 3×10−6 mol/l of 1, its leaf-opening activity lasted
for only 2 days. After that, the leaves became closed at
night again. This is because 1 is gradually hydrolyzed
into 3 within a few days in the plant body (Fig. 1). On
the other hand, leaf-opening activity of 2 lasted even
after 1 week. The leaves treated with 3×10−6 mol/l of 2
remained open until it withered and died after 2 weeks
(Fig. 2). Same results were obtained by using 4 and 5.
These results clearly showed that leaf closure is essential
for survival of this plant.
According to the mechanism shown in Fig. 1, it was
expected that structurally modified leaf-opening sub-
stance that cannot be hydrolyzed by b-glucosidase
would keep the leaf open constantly, and, thus, inhibit
the leaf-closure (causing ‘insomnia’). Potassium les-
pedezate (1) was isolated as a glucoside-type leaf open-
ing substance that is effective for the leaf of a
leguminous plant, Cassia mimosoides L.6 Structure–
activity relationship studies had shown that structural
modification on the sugar moiety of 1 caused no
decrease in bioactivity.6,8 Then, based on the structure
of 1, we designed and synthesized a potential leaf-
movement inhibitors (2, 4, and 5) that are expected not
to be hydrolyzed by b-glucosidase in the plant body
(Fig. 1).6,8 Leaf-movement inhibitors showed novel
bioactivities in bioassay; the leaves detached from the
The death of leaf is also attributable to potential toxic
feature of 2, 4, and 5. However, we have some experi-
mental proofs that they operate as leaf-movement
inhibitors, and not as toxins in the plant body. Strong
correlation was observed between leaf-opening activity
and death of plant leaf. When the leaves were treated
with analogs of 2 that did not show leaf-opening activ-
ity even at 1×10−3 mol/l, such as one with a reduced
double bond (6a and 6b)9 and another with phenolic
methyl ether (7)10 that were prepared from 11 (Scheme
Figure 2. The effect of a leaf-movement inhibitor (2) on the leaf of C. mimosoides. Each leaf was soaked in a 3×10−6 mol/l aqueous
solution of 2 or distilled water only for the control sample; from left, the leaves on the 1st, 4th, and 14th day and a control on
14th day at 9:00 pm. These experiments were carried out in a biotron under the following conditions: 12 h light/12 h dark, at
27°C, 50% humidity.

M. Ueda et al. / Tetrahedron Letters 43 (2002) 7545–7548
7547
the soybean, which belongs to the same genus as
them. However, the use of a leaf-movement inhibitor
as a herbicide could remove these weeds without dam-
aging the soybean and other vicinal organisms.
Our result gives an important clue for the historic
mystery, ‘Why does the leguminous plant sleep?’ We
showed that nyctinastic leaf-movement is essential for
the survival of leguminous plants by using synthetic
inhibitor of nyctinasty.
Acknowledgements
This work was supported by the Ministry of Educa-
tion, Science, Sports and Culture (Japan) for Grant-
in-Aid for Scientific Research (No. 1302467 and
12680598), Pioneering Research Project in Biotechnol-
ogy given by the Ministry of Agriculture, Forestry and
Fisheries, Kato Memorial Bioscience Foundation,
Naito Foundation, Kurata Foundation, and Moritani
Foundation.
Scheme 1. Chemical synthesis of analogs of 2 without leaf-
opening activity.
References
1. Schildknecht, H. Angew. Chem. 1983, 95, 689–704;
Angew. Chem., Int. Ed. Engl. 1983, 22, 695–710.
2. Darwin, C. The Power of Movement in Plants; John
Murray: London, 1880.
1), the leaves did not suffer any damage with them
and never withered and died even after 2 weeks.
Moreover, 2, 4, and 5 showed extremely specific bioac-
tivity to the leaf of C. mimosoides, that is a special
feature also observed in 1.6 And 2, 4, and 5 showed
no leaf-opening activity with leaves of other plants,
such as Mimosa pudica L., Albizzia julibrissin Durazz,
Aeschynomene indica L. and Phyllanthus urinaria L.,
even at 3×10−5 mol/l. Also, after 2 weeks, no death of
the leaf was observed about these plants. This specific
bioactivity cannot be interpreted if 2, 4, and 5 oper-
ated as some toxin in the plant body of C. mimosoides.
These results strongly suggested that 2, 4, and 5 oper-
ated as leaf-movement inhibitors in the plant body
and cause withering and death of C. mimosoides by
inhibiting leaf closure.
3. Schwintzer, C. R. Plant Physiol. 1971, 48, 203–207.
4. Enright, J. T. Oecologia 1982, 54, 253–259.
5. Bu¨nning, E.; Moser, I. Proc. Natl. Acad. Sci. USA 1969,
62, 1018–1022.
6. Ueda, M.; Yamamura, S. Angew. Chem. 2000, 1456–
1471; Angew. Chem., Int. Ed. 2000, 39, 1400–1414.
7. Ueda, M.; Shigemori, H.; Sata, N.; Yamamura, S. Phyto-
chemistry 2000, 53, 39–44.
8. Ueda, M.; Sawai, Y.; Yamamura, S. Tetrahedron Lett.
1999, 40, 3757–3760.
1
9. Compound 6a: H NMR (400 MHz, D2O, 30°C): l 7.25
(2H, d, J=8.3 Hz), 6.88 (2H, d, J=8.3 Hz), 4.43 (1H, t,
J=6.8 Hz), 4.37 (1H, d, J=7.3 Hz), 3.91 (1H, d, J=2.0
Hz), 3.75 (1H, dd, J=7.3, 11.1 Hz), 3.71 (1H, dd, J=4.8,
11.1 Hz), 3.62–3.56 (3H, m), 3.08 (1H, dd, J=5.8, 13.6
Hz), 3.05 (1H, dd, J=6.8, 13.6 Hz); 13C NMR (100
MHz, D2O, 30°C): l 180.0, 156.7, 133.4, 130.9, 117.8,
105.2, 82.4, 77.6, 75.1, 73.2, 72.3, 71.1, 63.3; IR (film): w
3365, 1716, 1615, 1516 cm−1; HRMS (negative FAB)
calcd. for C15H19O9 [M−K]−: 343.1026, found: 343.1049;
And also, these results suggest an important applica-
tion of the leaf-movement inhibitor as a potential
environment-friendly herbicide of extremely high spe-
cificity. A herbicide based on leaf-movement inhibitor
would enable complete selectivity to the target legumi-
nous weed from which the substance was isolated, and
have no effect on vicinal plants, insects, birds, animals,
and human beings. For example, soybean [Glycine
max (L.) Merr.] is the most important leguminous
crop, and genetically engineered soybean (Monsanto’s
Roundup Ready Soybeans) is widely cultivated in the
USA. Unfortunately, some leguminous weeds, such as
Sesbania exaltata Cory and Senna obtusifolia, are resis-
tant to Roundup and can grow in fields of genetically
engineered soybean after the treatment of Roundup.
These weeds cause serious trouble because no existing
herbicide can remove these weeds without damaging
1
[h]2D2=−10.9° (c 0.57 in H2O). Compound 6b: H NMR
(400 MHz, D2O, 30°C): l 7.24 (2H, d, J=8.3 Hz), 6.87
(2H, d, J=8.3 Hz), 4.65 (1H, t, J=6.3 Hz), 4.24 (1H, d,
J=7.8 Hz), 3.92 (1H, d, J=3.4 Hz), 3.77 (1H, dd, J=7.8,
11.2 Hz), 3.74 (1H, dd, J=4.3, 11.2 Hz), 3.70–3.61 (2H,
m), 3.57 (1H, dd, J=7.8, 9.8 Hz), 3.11 (1H, dd, J=5.9,
14.1 Hz), 3.06 (1H, dd, J=6.3, 14.1 Hz); 13C NMR (100
MHz, D2O, 30°C): l 179.8, 156.7, 133.4, 131.1, 117.7,
104.6, 81.7, 77.7, 75.2, 73.4, 72.3, 71.0, 63.4; IR (film): w
3365, 1721, 1597, 1516 cm−1; HRMS (negative FAB)
calcd. for C15H19O9 [M−K]−: 343.1026, found: 343.1026;
[h]2D2=+2.0° (c 0.70, H2O).

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M. Ueda et al. / Tetrahedron Letters 43 (2002) 7545–7548
1
10. Compound 7: H NMR (400 MHz, D2O, 30°C): l 7.65
(2H, d, J=8.8 Hz), 6.86 (2H, d, J=8.8 Hz), 6.60 (1H, s),
4.82 (1H, d, J=7.6 Hz), 3.75 (1H, d, J=3.6 Hz), 3.69
(3H, s), 3.64 (1H, dd, 8.0, 10.0 Hz), 3.57–3.43 (4H, m);
13C NMR (100 MHz, D2O, 30°C): l 172.4, 160.6,
147.1, 133.3, 128.3, 121.7, 115.7, 103.3, 77.3, 74.6,
73.1, 71.5, 70.3, 62.6; IR (film): w 3362, 1604, 1574, 1511
cm−1; HRMS (negative FAB) calcd. for C16H19O9 [M−
K]−: 355.1029, found: 355.1003; [h]2D2=+56.2° (c 0.79,
H2O).