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Structure-activity relationships of benzoxazolinones with respect to auxin-induced growth and auxin-binding protein
Pergamon
Pkycaknirrry.
Vol 37. No. 2 pp 297. MO. 1994 copyfighl 0 1994EJmna sdnwx Lid Primed anGruc Bntti. All nghu rumwd cm1 -9422/94 17.a) + 0.00
STRUCTURE-ACTIVITY RELATIONSHIPS OF BENZOXAZOLINONES WITH RESPECT TO AUXIN-INDUCED GROWTH AND AUXIN-BINDING PROTEIN MASAKOHOSHI-SAKODA,KENJI USUI, Kozo
ISHIZUKA,
SEIJI KOSEMURA,*
SHOSUKE YAMAMURA*
and
KOJI HASEGAWAt Institute of Applied Biochemistry. University of Tsukuba. Tsukuba, Ibataki 305. Japan; *Department of Chemistry, Faculty of Science and Technology, Keio Univenity.
(Receired in
reoisedform
Key Word Index-Zea mays; Gramineae; structure-activity relationships.
Abstract-The
structure-activity
relationships
antiauxin;
of the naturally
Hiyoshi,
Yokohama
223, Japan
26 April 1994)
auxin-binding
occurring
protein;
auxin-inhibiting
benzoxazolinone;
substance, 6-methoxy-2-
from maize shoots, and its artificial analogues with respect to auxin activity and membranebound auxin-binding protein, were studied. 6-Isobutoxy-2benzoxazolinone strongly inhibited auxin (I-naphthylacetic acid, NAA)-induced growth of etiolated maize coleoptile segments. 6-Isopropoxy-2benzoxazolinone, 6cthoxy-2benzoxazolinone and 6-methoxy-2benzoxazolinone showed significant inhibitory activity. However, 2benzoxazolinone and 6-benzyloxy-2-benzoxazolinone did not inhibit auxin-induced growth. On the basis of these data, it seemed reasonable to assume that the auxin-inhibitory activity was enhanced by increasing the side chain length at position C6 on the 2benzoxazolinone, although the attachment of a ring at C-6 destroyed activity. Competition by benzoxazolinones with an alkoxy group at C-6 with 3H-NAA at auxin-binding protein(s) isolated from endoplasmic reticulum (ER) membrane of maize shoots showed a positive correlation with their physiological effects. However, since the inhibitory activity of the benzoxazolinones for auxin-receptor(s) binding was small compared with their physiological activity, the benzoxazolinones may contribute to inhibition of auxin-induced growth through interference with other auxin-receptors. benzoxazolinone,
isolated
INTRODUCTION
Evidence that phototropism is caused by the light-induced appearance of a gradient of growth inhibitors of auxin action has been given for many plant organs [ 1-IO]. In radish, the growth inhibiting raphanusanins accumulate at the illuminated side of hypocotyls subjected to phototropic stimulation, and cause growth inhibition through interference with auxin-mediated microtubule orientation [ll]. Furthermore, it was shown that lateral application of these compounds caused differential growth, which resulted in bending toward the applied side [3]. Recently, Hasegawa et al. isolated an auxin-inhibiting substance, which increased in relation to light, from light-grown maize (Zea mays) shoots and identified its structure as 6-methoxy-2benzoxazolinone [ 123. This compound had been found in maize and wheat as an antifungal factor [13] and also in Scopario dulcis [14]. It was reported that in the intact plant the compound occurs as glucoside, from which the benzoxazolinone is released enzymically upon cell damage, e.g. by pathogenic attack or by homogenization [15-IrJ. It was
fAuthor
to whom correspondena
also reported that the content of 6-methoxy-2benzoxazolinone in green seedlings was many times greater than that of seedlings grown in the dark [l2, 18, 193. The results suggest that light may also act by releasing 6-methoxy-2benzoxazolinone from its glucoside in plant tissues. The range of known activities of the benzoxazolinone has substantially widened and the understanding of these activities increased. Ray et al. found a substance which has the property of modifying the binding affinity of the sites for NAA in etiolated maize shoots and suggested it may be a natural regulator of auxin activity [20]. Later it was isolated and identified as a mixture of 2benzoxazolinone and 6.7-dimethoxy-2benzoxazolinone [21]. In this paper, we report on the structure-activity relationships of benzoxazolinones with respect to auxin-induced growth and membrane-bound auxin-binding protein(s).
a
RO,
0
0 \ 1 >
should be addressed. 297
H
BOA YBOA IBOA IPBOA IBBOA BBOA
R=
R= R= R= R= R=
H CHJ CHsCH, (CH&CH (CH&CHCH, bwlqi
M.
298
I
toe5
1
lo4
HOSHI-SAKODA et al.
I
10-S
lo4
lo4
1oq3
Concentration, log (M) Fig. 1. Effect of benzoxazoiinones on the auxin-induced growth of etiotated maize coleoptile. Each value is the average of 20 measurement% bars indicate se. BOA, 2-benzoxazolinone; BBOA, 6-benzyloxy-2benzoxazohnone; MBOA, kmethoxy-2-benzoxazolinone; EBOA, 6-ethoxy-2-benzoxazolinone; IPBOA, 6-isopropoxy-2. benzoxazolinone; IBBOA, 6-isobutoxy-2benzoxazohnone.
RESULTS AND DISCUSSION
The biological activities of benzoxazolinones were tested in the maize coleoptile section test (Fig. 1). 2Benzoxazolinones with an alkoxy group at the C-6 position showed inhibitory activity, but 2benzoxazolin ones without a side chain or with a benzyl ring at C-6 did not. Among the benzoxazolinones with an alkoxy group, 6-isobutoxy-2henzoxazolinone showed the greatest inhibitory activity. It seems reasonable to assume that the auxin inhibition will beenhanced by increasing the length of the side chain at position C-6. Auxin-binding protein from maize shoot membranes has been found in the ER (site-I) {223 and its site(s) have a high affinity for NAA. When the auxin-binding protein was tested for 3H-NAA binding over a concentration range of 8 x 10-8-10-6 M a linear Scatchard plot was obtained (Fig. 2). The protein bound NAA with a K, of 2.59 x IO-’ M. The obtained K, value is in good agreement with those found for the site-1 specific auxin-binding protein [23.24]. The displacement of NAA by a range of benzoxazolinones is shown in Fig. 3. Inhibition by benzoxazolinones with an alkoxy group at the C-6 position of ‘H-NAA binding to auxin-binding protein showed a positive correlation with their physiological activity. However, the competitive activity of the benzoxazolinones in auxin-binding protein was rather small compared with their physiological activity (Fig. I). These results indicate the possibility of the presence of other auxinbinding protein(s) than ER-membrane-bound receptor(s) playing a role in cell extension and acting as a target for benzoxazolinones.
0
0
02
0‘
06
08
10
12
14
16
18 x-0 7
Bound
(M)
Fig. 2. Scatchard plot of the binding data of ‘H-NAA binding protein(s).
to auxin-
It can be concluded that benzoxazolinones with an alkoxy group at the C-6 position contribute to the inhibition of auxin-induced growth through competition with auxin at receptor sites that occur, at least partly, at ER membranes. EXPERIMENTAL
Materials. Syntheses of 6-alkoxy-2benzoxazolinones were done according to the procedure of refs [25,26]. 3Alkoxyphenol was dissolved in HOAc and was nitrated at 0” by the gradual addition of a coned soln of HNO, in
Structure-activity relationships of bcnzoxazolinoncs
299
120
100
‘r
60
60
60
40
40
IBBOA
20
Concentration, Fig. 3. Structure-activity
log (M)
relationship for inhibition of NAA binding. s.e. of data is included in each mark. Abbreviations as in Fig. 1.
HOAc. The reaction mixt. was stirred at room temp. for 3 hr, and then poured into Hz0 and extracted with EtOAc. The EtOAc soln was washed with satd aq. NaCl and dried (Na,SO,). Removal of solvent afforded an oil, which was purified by CC (silica gel, Katayama Chemicals K070, Japan) using hexane and EtOAc (3: l), giving a pale yellow oil of 5-alkoxy-2-nitrophenol. To 5-alkoxy2-nitrophenol in H,O, solid anhydrous Na dithionite was added gradually with stirring, and heated at 75” for 1.5 hr. The suspension became a clear, yellow soln which, after cooling, was neutralized with solid NaHCOS. After addition of EtOAc, the EtOAc soln was washed with satd aq. NaCl and then dried (Na,SO,). The organic layer was coned under red. pres. to leave an oil, which was purified by CC over silica gel using hexane-EtOAc (2 : 1) to afford an amorphous powder. The amine dissolved when tteated with 4 M HCI and immediately formed a dark purple soln. Removal of solvent under red. pres. gave a purple powder, amine hydrochloride. A soln of amine hydrochloride and urea in 1,3-butanediol was heated at 170” for 2 hr with stirring The reaction mixt. was diluted with Hz0 and then extracted with EtOAc. The EtOAc soln was washed with satd NaCl and then dried (Na,SO,). Removal of solvent afforded an oil, which was sepd by prep. TLC (Kiesel gel 60 PFzs,, 1 mm) using hexane-EtOAc (1: 1) to give an amorphous powder of benzoxazolinone. Bioassay. Ten 4.5mm sections of 4&y-old etiolated maize (Zea muys L. cv Honey Bantam) coleoptiles were incubated in 1% sucrose soln (pH 5.6) containing lo- ’ M NAA and various concns of benzoxazolinones at 25” for 18 hr. After incubation, the length of each section was measured. Membrane preparation and solubilization. Microsomal membranes were prepd from 4day-old etiolated coleop tiles and enclosed leaf rolls of maize as described in ref.
[27]. The membrane pellet was washed with cold Me&O and dried in mcuo. The membranes were homogenized in resuspension buffer [lo mM Na citrate-citric acid, 5 mM MgCI,, 0.2 mM phenylmethylsulphonyl fluoride (PMSF, pH 5.5)]. After centrifugation at 10000 g for 20 min the supematant was passed through a PD-10 column (Pharmacia LKB) equilibrated with resuspension buffer minus PMSF, giving the solubilized auxin-binding protein(s). Auxin-binding assays. Each test compound and 5 x 10-a M ‘H-NAA (0.18 TBq mmol-‘) were added to 0.5 ml of solubilized auxin-binding protein(s) (ca 0.4 mg of protein ml-‘) in binding buffer (10 mM Na citrate-citric acid, 5 mM MgCI,, pH 5.5) and incubated for 5 min at 4”. Incubation was stopped by addition of 1 ml of satd (NH&SO,. Samples were centrifuged for 30 min at 25 000 g, very carefully decanted, dissolved in 100 ~1 of 0.1% SDS and whole tubes analysed for radioactivity in a liquid scintillation counter [28]. Acknowledgements-We thank Sumitomo Chemicals for the supply of ‘H-NAA, Dr N. Mito (Agricultural Science Laboratory, Sumitomo Chemicals) for his valuable suggestions and Prof. 1. Bruinsma, The Netherlands for his valuable comments. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan. The senior author thanks the Japanese Society for the Promotion of Science for a fellowship. REFERENCES
Bruinsma, J., Karssen, C. M., Benschop, M. and Van Dort, J. B. (1975) J. Exp. Botany 26, 411. Franssen, J. M. and Bruinsma, J. (1981) Planta 151, 365. Noguchi, H., Nishitani, K., Bruinsma, J. and Hasegawa, K. (1986) Plant Physiol. 81, 980.
M HOSHI-SAKODAer al
300 4. Feyerabend,
Plum.
M. and Weiler,
E. W. (1988)
Physiol.
17. Hietala, Stand.
74. 185.
5. Sakoda,
K. (1989) Physiol.
M. and Hasegawa,
Planf.
18. Wilkins, ,4ppl.
76, 240. 6. Bruinsma. Botany
J. and Hasegawa.
K.
(1989) Environ.
Exp.
7. Hasegawa. PIonro
K.. Sakoda.
M. and Bruinsma.
J. (1989)
K. (1990) Physiol.
Plow.
79. 700.
21.
K. (1991) Physiol.
Planr.
23.
555. K. and Yamada.
K. (1992) J. Planr
24.
Physiol.
Plant.
12. Hasegawa.
S. and
Yamamura.
M., Mizutani, S. (1992)
P. K. and
(1956) Suomen Kemisrilehri
B 29, 143.
Scund.
Phyro-
M. (1976)
Wahlroos.
Phprochemisrry
d.
25.
Stand.
0. and Vtrtanen.
A. I. (1959) Acra
Chem.
26. 27.
Biol.
A. I. and Hietala.
28.
P. K. (1960) Acra
Chem.
Chem.
D.. Bauw, G., M., Klambt. Molec.
Biol.
39, 683.
Richey, J. D.. Caskey, A. L. and BeMiller. Brol. Chem.
Shtmomura, Hajek,
J. N. (1976)
40. 2413.
S.. Sotobayashi. 99.
T., Futai. M. and Fukui.
1513.
K. and Jacobsen, H.-J. (1987) in Plant
mone Receprors
13. 1906. 14,499.
J., lobler.
Rickey, J. D., Scism. A. J.. Caskey, A. L. and &Miller,
Agric. 15,
260,
J. 8. 2453.
T. (1986) J. Biochem.
16. Virtanen,
D. (1985) J. Biol. Chem.
Hesse, T.. Feldwisch, J.. Balshusemann.
Org.
J.,
1997. 15. Wahlroos.
K. and Russo. V. E. R. (1972)
Lobler. M. and Klambt.
J. N. (1975) Ayric.
A. I., Hietala,
C. and Chen.
R.. Thomson, 107. 325.
D.. Schell. J. and Palme, K. (1989) Eur.
31, 3673.
13. Virtanen.
U. and Hertel. R. (1977) Plonr
59. 357.
Puype, M., Vandekerckhove.
K. (1992)
84, 509.
K., Togo, S., Urashima,
Kosemura, chemisrq,
K. and Ishizuka.
c).
69, 486.
9848.
Phys-
iol. 139, 455.
I I. Sakoda, M., Hasegawa.
P. K. and Wahlroos.
Biophys.
Venis. M. A. and Watson, P. J. (1978) Planta 142, 103. Planra
81.
Biochem.
Ray, P. M., Dohrmann.
22. Hertel.
9. Togo. S. and Hasegawa,
14. Chcn.
20.
Physiol.
8. Bruinsma. 1. and Hasegawa,
IO. Hasegawa.
78. 337. A. I., Hietala.
(1957) Arch.
178, 540.
Chem.
H.. Burden, R. S. and Wain, R. L. (1974) Ann.
Biol.
19. Virtanen.
29. 25.
A. I. (1960) Acra
P. K. and Virtanen, 14, 502.
Series, Springer.
(Klambt. Berlin.
D.. ed.), p. 265. NATO
HorASI
Pkycaknirrry.
Vol 37. No. 2 pp 297. MO. 1994 copyfighl 0 1994EJmna sdnwx Lid Primed anGruc Bntti. All nghu rumwd cm1 -9422/94 17.a) + 0.00
STRUCTURE-ACTIVITY RELATIONSHIPS OF BENZOXAZOLINONES WITH RESPECT TO AUXIN-INDUCED GROWTH AND AUXIN-BINDING PROTEIN MASAKOHOSHI-SAKODA,KENJI USUI, Kozo
ISHIZUKA,
SEIJI KOSEMURA,*
SHOSUKE YAMAMURA*
and
KOJI HASEGAWAt Institute of Applied Biochemistry. University of Tsukuba. Tsukuba, Ibataki 305. Japan; *Department of Chemistry, Faculty of Science and Technology, Keio Univenity.
(Receired in
reoisedform
Key Word Index-Zea mays; Gramineae; structure-activity relationships.
Abstract-The
structure-activity
relationships
antiauxin;
of the naturally
Hiyoshi,
Yokohama
223, Japan
26 April 1994)
auxin-binding
occurring
protein;
auxin-inhibiting
benzoxazolinone;
substance, 6-methoxy-2-
from maize shoots, and its artificial analogues with respect to auxin activity and membranebound auxin-binding protein, were studied. 6-Isobutoxy-2benzoxazolinone strongly inhibited auxin (I-naphthylacetic acid, NAA)-induced growth of etiolated maize coleoptile segments. 6-Isopropoxy-2benzoxazolinone, 6cthoxy-2benzoxazolinone and 6-methoxy-2benzoxazolinone showed significant inhibitory activity. However, 2benzoxazolinone and 6-benzyloxy-2-benzoxazolinone did not inhibit auxin-induced growth. On the basis of these data, it seemed reasonable to assume that the auxin-inhibitory activity was enhanced by increasing the side chain length at position C6 on the 2benzoxazolinone, although the attachment of a ring at C-6 destroyed activity. Competition by benzoxazolinones with an alkoxy group at C-6 with 3H-NAA at auxin-binding protein(s) isolated from endoplasmic reticulum (ER) membrane of maize shoots showed a positive correlation with their physiological effects. However, since the inhibitory activity of the benzoxazolinones for auxin-receptor(s) binding was small compared with their physiological activity, the benzoxazolinones may contribute to inhibition of auxin-induced growth through interference with other auxin-receptors. benzoxazolinone,
isolated
INTRODUCTION
Evidence that phototropism is caused by the light-induced appearance of a gradient of growth inhibitors of auxin action has been given for many plant organs [ 1-IO]. In radish, the growth inhibiting raphanusanins accumulate at the illuminated side of hypocotyls subjected to phototropic stimulation, and cause growth inhibition through interference with auxin-mediated microtubule orientation [ll]. Furthermore, it was shown that lateral application of these compounds caused differential growth, which resulted in bending toward the applied side [3]. Recently, Hasegawa et al. isolated an auxin-inhibiting substance, which increased in relation to light, from light-grown maize (Zea mays) shoots and identified its structure as 6-methoxy-2benzoxazolinone [ 123. This compound had been found in maize and wheat as an antifungal factor [13] and also in Scopario dulcis [14]. It was reported that in the intact plant the compound occurs as glucoside, from which the benzoxazolinone is released enzymically upon cell damage, e.g. by pathogenic attack or by homogenization [15-IrJ. It was
fAuthor
to whom correspondena
also reported that the content of 6-methoxy-2benzoxazolinone in green seedlings was many times greater than that of seedlings grown in the dark [l2, 18, 193. The results suggest that light may also act by releasing 6-methoxy-2benzoxazolinone from its glucoside in plant tissues. The range of known activities of the benzoxazolinone has substantially widened and the understanding of these activities increased. Ray et al. found a substance which has the property of modifying the binding affinity of the sites for NAA in etiolated maize shoots and suggested it may be a natural regulator of auxin activity [20]. Later it was isolated and identified as a mixture of 2benzoxazolinone and 6.7-dimethoxy-2benzoxazolinone [21]. In this paper, we report on the structure-activity relationships of benzoxazolinones with respect to auxin-induced growth and membrane-bound auxin-binding protein(s).
a
RO,
0
0 \ 1 >
should be addressed. 297
H
BOA YBOA IBOA IPBOA IBBOA BBOA
R=
R= R= R= R= R=
H CHJ CHsCH, (CH&CH (CH&CHCH, bwlqi
M.
298
I
toe5
1
lo4
HOSHI-SAKODA et al.
I
10-S
lo4
lo4
1oq3
Concentration, log (M) Fig. 1. Effect of benzoxazoiinones on the auxin-induced growth of etiotated maize coleoptile. Each value is the average of 20 measurement% bars indicate se. BOA, 2-benzoxazolinone; BBOA, 6-benzyloxy-2benzoxazohnone; MBOA, kmethoxy-2-benzoxazolinone; EBOA, 6-ethoxy-2-benzoxazolinone; IPBOA, 6-isopropoxy-2. benzoxazolinone; IBBOA, 6-isobutoxy-2benzoxazohnone.
RESULTS AND DISCUSSION
The biological activities of benzoxazolinones were tested in the maize coleoptile section test (Fig. 1). 2Benzoxazolinones with an alkoxy group at the C-6 position showed inhibitory activity, but 2benzoxazolin ones without a side chain or with a benzyl ring at C-6 did not. Among the benzoxazolinones with an alkoxy group, 6-isobutoxy-2henzoxazolinone showed the greatest inhibitory activity. It seems reasonable to assume that the auxin inhibition will beenhanced by increasing the length of the side chain at position C-6. Auxin-binding protein from maize shoot membranes has been found in the ER (site-I) {223 and its site(s) have a high affinity for NAA. When the auxin-binding protein was tested for 3H-NAA binding over a concentration range of 8 x 10-8-10-6 M a linear Scatchard plot was obtained (Fig. 2). The protein bound NAA with a K, of 2.59 x IO-’ M. The obtained K, value is in good agreement with those found for the site-1 specific auxin-binding protein [23.24]. The displacement of NAA by a range of benzoxazolinones is shown in Fig. 3. Inhibition by benzoxazolinones with an alkoxy group at the C-6 position of ‘H-NAA binding to auxin-binding protein showed a positive correlation with their physiological activity. However, the competitive activity of the benzoxazolinones in auxin-binding protein was rather small compared with their physiological activity (Fig. I). These results indicate the possibility of the presence of other auxinbinding protein(s) than ER-membrane-bound receptor(s) playing a role in cell extension and acting as a target for benzoxazolinones.
0
0
02
0‘
06
08
10
12
14
16
18 x-0 7
Bound
(M)
Fig. 2. Scatchard plot of the binding data of ‘H-NAA binding protein(s).
to auxin-
It can be concluded that benzoxazolinones with an alkoxy group at the C-6 position contribute to the inhibition of auxin-induced growth through competition with auxin at receptor sites that occur, at least partly, at ER membranes. EXPERIMENTAL
Materials. Syntheses of 6-alkoxy-2benzoxazolinones were done according to the procedure of refs [25,26]. 3Alkoxyphenol was dissolved in HOAc and was nitrated at 0” by the gradual addition of a coned soln of HNO, in
Structure-activity relationships of bcnzoxazolinoncs
299
120
100
‘r
60
60
60
40
40
IBBOA
20
Concentration, Fig. 3. Structure-activity
log (M)
relationship for inhibition of NAA binding. s.e. of data is included in each mark. Abbreviations as in Fig. 1.
HOAc. The reaction mixt. was stirred at room temp. for 3 hr, and then poured into Hz0 and extracted with EtOAc. The EtOAc soln was washed with satd aq. NaCl and dried (Na,SO,). Removal of solvent afforded an oil, which was purified by CC (silica gel, Katayama Chemicals K070, Japan) using hexane and EtOAc (3: l), giving a pale yellow oil of 5-alkoxy-2-nitrophenol. To 5-alkoxy2-nitrophenol in H,O, solid anhydrous Na dithionite was added gradually with stirring, and heated at 75” for 1.5 hr. The suspension became a clear, yellow soln which, after cooling, was neutralized with solid NaHCOS. After addition of EtOAc, the EtOAc soln was washed with satd aq. NaCl and then dried (Na,SO,). The organic layer was coned under red. pres. to leave an oil, which was purified by CC over silica gel using hexane-EtOAc (2 : 1) to afford an amorphous powder. The amine dissolved when tteated with 4 M HCI and immediately formed a dark purple soln. Removal of solvent under red. pres. gave a purple powder, amine hydrochloride. A soln of amine hydrochloride and urea in 1,3-butanediol was heated at 170” for 2 hr with stirring The reaction mixt. was diluted with Hz0 and then extracted with EtOAc. The EtOAc soln was washed with satd NaCl and then dried (Na,SO,). Removal of solvent afforded an oil, which was sepd by prep. TLC (Kiesel gel 60 PFzs,, 1 mm) using hexane-EtOAc (1: 1) to give an amorphous powder of benzoxazolinone. Bioassay. Ten 4.5mm sections of 4&y-old etiolated maize (Zea muys L. cv Honey Bantam) coleoptiles were incubated in 1% sucrose soln (pH 5.6) containing lo- ’ M NAA and various concns of benzoxazolinones at 25” for 18 hr. After incubation, the length of each section was measured. Membrane preparation and solubilization. Microsomal membranes were prepd from 4day-old etiolated coleop tiles and enclosed leaf rolls of maize as described in ref.
[27]. The membrane pellet was washed with cold Me&O and dried in mcuo. The membranes were homogenized in resuspension buffer [lo mM Na citrate-citric acid, 5 mM MgCI,, 0.2 mM phenylmethylsulphonyl fluoride (PMSF, pH 5.5)]. After centrifugation at 10000 g for 20 min the supematant was passed through a PD-10 column (Pharmacia LKB) equilibrated with resuspension buffer minus PMSF, giving the solubilized auxin-binding protein(s). Auxin-binding assays. Each test compound and 5 x 10-a M ‘H-NAA (0.18 TBq mmol-‘) were added to 0.5 ml of solubilized auxin-binding protein(s) (ca 0.4 mg of protein ml-‘) in binding buffer (10 mM Na citrate-citric acid, 5 mM MgCI,, pH 5.5) and incubated for 5 min at 4”. Incubation was stopped by addition of 1 ml of satd (NH&SO,. Samples were centrifuged for 30 min at 25 000 g, very carefully decanted, dissolved in 100 ~1 of 0.1% SDS and whole tubes analysed for radioactivity in a liquid scintillation counter [28]. Acknowledgements-We thank Sumitomo Chemicals for the supply of ‘H-NAA, Dr N. Mito (Agricultural Science Laboratory, Sumitomo Chemicals) for his valuable suggestions and Prof. 1. Bruinsma, The Netherlands for his valuable comments. This work was supported in part by grants from the Ministry of Education, Science and Culture, Japan. The senior author thanks the Japanese Society for the Promotion of Science for a fellowship. REFERENCES
Bruinsma, J., Karssen, C. M., Benschop, M. and Van Dort, J. B. (1975) J. Exp. Botany 26, 411. Franssen, J. M. and Bruinsma, J. (1981) Planta 151, 365. Noguchi, H., Nishitani, K., Bruinsma, J. and Hasegawa, K. (1986) Plant Physiol. 81, 980.
M HOSHI-SAKODAer al
300 4. Feyerabend,
Plum.
M. and Weiler,
E. W. (1988)
Physiol.
17. Hietala, Stand.
74. 185.
5. Sakoda,
K. (1989) Physiol.
M. and Hasegawa,
Planf.
18. Wilkins, ,4ppl.
76, 240. 6. Bruinsma. Botany
J. and Hasegawa.
K.
(1989) Environ.
Exp.
7. Hasegawa. PIonro
K.. Sakoda.
M. and Bruinsma.
J. (1989)
K. (1990) Physiol.
Plow.
79. 700.
21.
K. (1991) Physiol.
Planr.
23.
555. K. and Yamada.
K. (1992) J. Planr
24.
Physiol.
Plant.
12. Hasegawa.
S. and
Yamamura.
M., Mizutani, S. (1992)
P. K. and
(1956) Suomen Kemisrilehri
B 29, 143.
Scund.
Phyro-
M. (1976)
Wahlroos.
Phprochemisrry
d.
25.
Stand.
0. and Vtrtanen.
A. I. (1959) Acra
Chem.
26. 27.
Biol.
A. I. and Hietala.
28.
P. K. (1960) Acra
Chem.
Chem.
D.. Bauw, G., M., Klambt. Molec.
Biol.
39, 683.
Richey, J. D.. Caskey, A. L. and BeMiller. Brol. Chem.
Shtmomura, Hajek,
J. N. (1976)
40. 2413.
S.. Sotobayashi. 99.
T., Futai. M. and Fukui.
1513.
K. and Jacobsen, H.-J. (1987) in Plant
mone Receprors
13. 1906. 14,499.
J., lobler.
Rickey, J. D., Scism. A. J.. Caskey, A. L. and &Miller,
Agric. 15,
260,
J. 8. 2453.
T. (1986) J. Biochem.
16. Virtanen,
D. (1985) J. Biol. Chem.
Hesse, T.. Feldwisch, J.. Balshusemann.
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