bk-1991-0474.ch002

Chapter 2 Brassinosteroids in Leaves of Distylium racemosum Sieb. et Zucc. The Beginning of Brassinosteroid Research in Japan 1

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Hiroshi Abe and Shingo Marumo Downloaded by UNIV OF ARIZONA on January 8, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch002

1

Department of Plant Protection, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan Department of Agricultural Chemistry, Nagoya University, Chikusa, Nagoya 464, Japan 2

The first preliminary report of brassinosteroids in Japan was released in 1968 under the title " The Presence of Novel Plant Growth Regulators in Leaves of Distylium racemosum Sieb. et Zucc. " two years before the first report on the development of b r a s s i n s , the crude brassinolide, by Mitchell et al. in Nature(1). From 430 kg of leaves 751 μg of Distylium factor A , 50 μg of A and 236 μg of B were isolated and all showed much higher activity than indole-3-acetic acid [IAA] on a rice-lamina inclination bioassay. However, the very minute amounts precluded any chemical studies at that time. Just after the structure of brassinolide was announced by the USDA group in 1979, (22S, 23S)-24-homobrassinolide and brassinolide were synthesized by Japanese chemists, and their biological activity was examined by the rice lamina inclination (or joint) test. Both compounds showed activity levels ten thousand times stronger than the activity of IAA, demonstrating that this bioassay was exclusively specific for brassinosteroids and was not contaminated by other plant hormones. Distylium factors were reinvestigated, and A was identified as a mixture of brassinone and castasterone and B as a mixture of brassinolide and 28-norbrassinolide. 1

2

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Distylium racemosum S i e b . et Z u c c , k n o w n as " I s u n o k i " i n Japanese, is a n e v e r g r e e n t r e e b e l o n g i n g to the family Hamamelidaceae. It i s w e l l k n o w n t h a t s e v e r a l v a r i e t i e s of a p h i d s form i n s e c t g a l l s o n t h e y o u n g , f r e s h l e a v e s o r b u d s of t h i s t r e e . The a p h i d , Neothoracaphis yanonis, a t t a c k s t h e small, y o u n g l e a v e s o n t h e t r e e . T h e a t t a c k e d leaf soon b e g i n s to s w e l l a n d to form a q u i t e small g a l l ( g a l l diameter, l-2mm) i n S p r i n g . I n M a y , t h e i n s e c t g a l l o n t h e leaf r a p i d l y grows a n d enlarges (gall diameter

0097-6156/91/0474-0018$06.00/0 © 1991 American Chemical Society In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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ABE & MARUMO

Research in Japan

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Downloaded by UNIV OF ARIZONA on January 8, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch002

a b o u t 1 cm). Distylium f a c t o r r e s e a r c h was b e g a n i n a n e f f o r t to identify the substance which induces the abnormal growth, swelling and expansion observed d u r i n g insect gall formation. P r o f . M u n a k a t a of N a g o y a U n i v e r s i t y f o u n d a n a c t i v e s u b s t a n c e i n t h e n e u t r a l f r a c t i o n of methanol e x t r a c t s of t h e i n s e c t g a l l s w h e n b i o a s s a y e d b y means of a r i c e - l a m i n a i n c l i n a t i o n (or j o i n t ) t e s t [ L J T ] , w h i c h h a d b e e n e s t a b l i s h e d as a n a u x i n b i o a s s a y i n 1965 b y P r o f . M a e d a of N a g o y a U n i v e r s i t y ( i 5 . I n 1966, we c a r r i e d o u t f u r t h e r investigations for the p u r i f i c a t i o n , i s o l a t i o n a n d e l u c i d a t i o n of t h e c h e m i c a l p r o p e r t i e s of the active substance.

Distylium F a c t o r s : N o v e l P l a n t Growth Regulators i n F r e s h of Distylium racemosum S i e b . et Z u c c .

Leaves

A preliminary experiment revealed the presence of t h e a c t i v e s u b s t a n c e not o n l y i n the i n s e c t g a l l s o n t h e l e a v e s b u t a l s o i n f r e s h h e a l t h y l e a v e s of Distylium racemosum. F r e s h h e a l t h y l e a v e s (430 k g ) of the plant, therefore, were collected i n the s u b u r b s of A n j o c i t y at t h e e n d of M a y , 1966. M e t h a n o l e x t r a c t s of t h e m a t e r i a l were s e p a r a t e d b y c o n v e n t i o n a l t e c h n i q u e s to g i v e a n ether-soluble neutral substance having fairly strong activity i n t h e L J T . T h e n e u t r a l s u b s t a n c e was c h r o m a t o g r a p h e d on a s i l i c i c a c i d c o l u m n ( / 7 - h e x a n e - E t O A c - E t O H ) , from w h i c h Distylium f a c t o r A f r a c t i o n , i n c l u d i n g A i a n d A2, was e l u t e d w i t h 80% E t O A c - / ? hexane, a n d t h e n Distylium f a c t o r B w i t h 100% E t O A c . E a c h f r a c t i o n was f u r t h e r m o r e purified repeatedly b y Sephadex LH-20 gel filtration column chromatography(EtOH), Silica gel T L C (Kieselgel H, EtOAc), and alumina column chromatography (EtOAc-EtOH) a f f o r d i n g t h r e e k i n d s of a c t i v e s u b s t a n c e s . A l l t h r e e w e r e d e t e c t e d o n a t h i n - l a y e r c h r o m a t o g r a m as a p u r p l e s p o t u n d e r u l t r a v i o l e t l i g h t u p o n h e a t i n g w i t h 0.5% v a n i l l i n - H 2 S 0 4 . The amount of Distylium f a c t o r s A i , A2, a n d B , named a f t e r t h e i r b o t a n i c a l s o u r c e , was 751, 50 a n d 236 11 g , r e s p e c t i v e l y . T h e Rf v a l u e s of Distylium f a c t o r s o n s i l i c a g e l T L C d e v e l o p e d w i t h EtOAc were: A i , 0.35; A2, 0.55; B, 0.13. The v a l u e s of P P C d e v e l o p e d w i t h isoPrOH/NH40H/H20 (8:1:1, v / v ) w e r e : A i , A2, B , 0.9-1.0. T h e s e f a c t o r s h a d c l e a r l y d i f f e r e n t Rf v a l u e s from t h e k n o w n p h y t o h o r m o n e s , were n e g a t i v e f o r E h l r i c h a n d S a l k o w s k y r e a g e n t s , a n d w e r e d e t e c t a b l e as a p u r p l e s p o t u n d e r u l t r a v i o l e t l i g h t a f t e r s p r a y i n g w i t h H 2 S O 4 c o n t a i n i n g 0.5% v a n i l l i n , s u g g e s t i n g that t h e y are n o n - i n d o l y l compounds. The amounts i s o l a t e d were too small to t e c h n i c a l l y p e r f o r m t h e i r structural d e t e r m i n a t i o n b y means of s p e c t r o s c o p i c a n a l y s i s at t h a t time, so i t was d e c i d e d to h a l t t h e s t u d i e s a t t h i s p o i n t . B i o l o g i c a l a c t i v i t i e s of Distylium f a c t o r s were i n v e s t i g a t e d using several known bioassays for plant hormones. Distylium f a c t o r s B a n d A i were f o u n d to e x h i b i t m u c h h i g h e r a c t i v i t y t h a n IAA i n t h e L J T ; t h a t i s , t h e same i n c l i n a t i o n s of l a m i n a j o i n t s e c t i o n s were o b s e r v e d w h e n the f a c t o r s were a p p l i e d at c o n c e n t r a t i o n s of one h u n d r e d t h a n d one t h i r t i e t h , r e s p e c t i v e l y . These factors also d i s p l a y e d s t r o n g e r elongation than IAA i n the

In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS

A vena f i r s t - i n t e r n o d e t e s t , t h e a c t i v i t y of B was a b o u t 10 times h i g h e r , w h e r e a s t h e y s h o w e d l o w e r a c t i v i t y i n t h e Avena c o l e o p t i l e s e c t i o n t e s t at a c o n c e n t r a t i o n of 0.01 ppm, a n d w e r e n e g a t i v e f o r t h e Avena c u r v a t u r e t e s t at a c o n c e n t r a t i o n of 5 p p m . T h e l e t t e r is a c l a s s i c a l bioassay for auxin detection. The c y t o k i n i n a c t i v i t y of B was a l s o t e s t e d a n d r e c o g n i z e d i n t h e r a d i s h leaf e x p a n s i o n t e s t a n d t o b a c c o p i t h c a l l u s t e s t . The a c t i v i t y of B was n e a r l y e q u a l to t h a t of k i n e t i n at a c o n c e n t r a t i o n of 0.1 p p m . GA a c t i v i t y was a l s o examined, b u t was not d e t e c t e d i n either the rice s e e d l i n g or the d w a r f maize s e e d l i n g e l o n g a t i o n t e s t s . As d e s c r i b e d above, the results of t e s t s c l e a r l y i n d i c a t e d t h a t Distylium f a c t o r s w e r e new t y p e s of p l a n t g r o w t h r e g u l a t o r s w h i c h c o m b i n e d some of t h e a t t r i b u t e s of a u x i n a c t i v i t y w i t h t h o s e of c y t o k i n i n a c t i v i t y a n d w h i c h h a d a b r o a d s p e c t r u m of p l a n t physiological activity. The u n i v e r s a l i t y of d i s t r i b u t i o n of Distylium f a c t o r s A a n d B i n p l a n t s was t e s t e d u s i n g methanol e x t r a c t s of f o u r t e e n p l a n t s p e c i e s . P l a n t s s u r v e y e d were Disanthus cercidifolia (leaves), Hamamelis japonica (leaves), Corylopsis glabrescens (leaves), Pittosporum tobira ( l e a v e s ) , Camellia sasanqua (leaves), Thea sinensis ( l e a v e s ) , Cocculus trilobus ( l e a v e s ) , Viburnum awabuki ( l e a v e s ) , Phaseolus vulgaris (shoots), Pisum sativum (seeds), Vicia faba (seeds), Dolichos lablab (seeds), Glycine max (seeds) a n d Arachis hypogaea ( s e e d s ) . A f t e r p a r t i t i o n of the neutral s u b s t a n c e s , s e p a r a t e d from methanol e x t r a c t s , b e t w e e n C H 3 C N a n d /2-hexane, t h e C H 3 C N - s o l u b l e p o r t i o n s were c h a r g e d onto a n AI2O3 c o l u m n . Distylium f a c t o r A was e l u t e d w i t h 20% EtOH i n EtOAc a n d t h e n Distylium f a c t o r B w i t h 40% EtOH i n E t O A c . B o t h f r a c t i o n s w e r e a p p l i e d to the L J T . Distylium f a c t o r s A a n d B w e r e c o n f i r m e d to be p r e s e n t n o t o n l y i n Distylium racemosum^ b u t w e r e f o u n d to be d i s t r i b u t e d w i d e l y a c r o s s a v a r i e t y of h i g h e r p l a n t s s u r v e y e d . A s mentioned a b o v e , t h e r e s e a r c h i n t o Distylium f a c t o r s has p r o v i d e d the f o l l o w i n g s i g n i f i c a n t i n f o r m a t i o n . Distylium f a c t o r s : (1) a r e d e t e c t a b l e as a p u r p l e s p o t u n d e r U V l i g h t a f t e r s p r a y i n g 0.5% v a n i l l i n - s u l f u r i c a c i d a n d t h e n h e a t i n g ; (2) s h o w s s e v e r a l h u n d r e d times h i g h e r a c t i v i t y t h a n IAA i n L J T ; (3) p o s s e s s e s a b r o a d s p e c t r u m of b i o l o g i c a l a c t i v i t i e s c o m p a r e d w i t h the k n o w n p h y t o h o r m o n e s ; a n d (4) a r e d i s t r i b u t e d w i d e l y i n t h e p l a n t k i n g d o m . We r e p o r t e d t h e s e r e s u l t s i n Agric. Biol. Chem. i n 1968 (3).

P u r i f i c a t i o n a n d I s o l a t i o n of Corn F a c t o r from C o r n Germ O i l I n r e l a t i o n to Distylium f a c t o r s research in Distylium racemosum l e a v e s , P r o f . M u n a k a t a has further studied the p r o v i s i o n a l l y named " Corn f a c t o r " from c o r n germ oil(4). He has examined many p l a n t o i l s i n a n e f f o r t to o b t a i n a b e t t e r s o u r c e of novel plant g r o w t h r e g u l a t o r s , i n c l u d i n g coconut o i l , c o r n germ o i l , c o t t o n seed o i l , l i n s e e d o i l , o l i v e o i l , p e a n u t o i l , p o p p y o i l , r a p e o i l , sesame o i l , s o y a o i l , a n d t u r p e n t i n e o i l , a n d found that c o r n g e r m o i l s h o w e d a most r e m a r k a b l e a c t i v i t y i n t h e L J T . B a s e d


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on t h e p u r i f i c a t i o n p r o c e d u r e f o r Distylium f a c t o r , a n active s u b s t a n c e named Corn f a c t o r was e x t r a c t e d a n d s e p a r a t e d from t h e n e u t r a l f r a c t i o n of the o i l a n d f i n a l l y i s o l a t e d i n the form of needle c r y s t a l s w i t h a y i e l d of 2 mg. T h e a c t i v i t y of Corn f a c t o r was a b o u t 2500 times h i g h e r t h a n t h a t of I A A i n the L J T , b u t was s i m i l a r to IAA i n the Avena f i r s t i n t e r n o d e t e s t . F u r t h e r s t u d i e s on t h e c h e m i c a l s t r u c t u r e of Corn f a c t o r c o u l d not be c o n d u c t e d b e c a u s e of i n s u f f i c i e n t y i e l d .

The Rice L a m i n a Brassinosteroids

Inclination

as

a

Specific

Bioassay

for


The i s o l a t i o n a n d s t r u c t u r a l d e t e r m i n a t i o n of b r a s s i n o l i d e [BR] was r e p o r t e d i n N a t u r e b y D r . N . B . M a n d a v a a n d c o - w o r k e r s at t h e USDA i n 1979(5), a n d was s u b s e q u e n t l y a n n o u n c e d i n Chem. & Eng. News(6>). The s t e r o i d a l s k e l e t o n of t h e c h e m i c a l s t r u c t u r e of BR p r o m p t e d u s to examine the effect o n the L J T . ( 2 2 5 , 2 3 5 ) h o m o b r a s s i n o l i d e was s y n t h e s i z e d b y M o r i i n 1980(7) at our r e q u e s t j u s t a f t e r the s t r u c t u r a l d e t e r m i n a t i o n of BR. BR i t s e l f was also p r o d u c e d b y I k e k a w a a n d h i s c o - w o r k e r s t h e same y e a r ( 6 ) . Wada, a c o - w o r k e r i n o u r g r o u p , t e s t e d t h e a c t i v i t y of BR a n d h o m o b r a s s i n o l i d e on r i c e lamina i n c l i n a t i o n a n d f o u n d t h a t b o t h c o m p o u n d s d r a m a t i c a l l y i n d u c e d t h e i n c l i n a t i o n of laminae (see T a b l e I) as e x p e c t e d ( S ) . These f i n d i n g s s t r o n g l y suggested

T a b l e I. E f f e c t of b r a s s i n o l i d e , h o m o b r a s s i n o l i d e * a n d l a m i n a i n c l i n a t i o n of r i c e s e e d l i n g s

IAA on

A n g l e degrees between laminae and sheaths ( ± s t a n d a r d Cone (//g/ml) O(control) 50 10 5 1 0.5 0.1 0.05 0.01 0.005 0.001 0.0005 0.0001

IAA 91 124 90 101 85

Brassinolide

error)

Homobrassinolide

± 6.6 ± 8.6 ± 1.7 ± 12.4 ± 9.0 161 169 158 156 163 150 143 116

± 9.1 ± 11.6 ± 8.5 ± 9.9 ± 7.2 ± 16.2 ± 11.1 ± 9.8

the

175 159 159 155 151 114 123 112

± ± ± ± ± ± ± ±

8.3 9.7 9.8 12.9 11.0 12.7 6.6 17.1

a

T h i s compound i s a s t e r e o i s o m e r i c m i x t u r e o f 2 a , 3 a ,227?,23#and 2 a , 3 a , 2 2 5 , 2 3 5 - t e t r a h y d r o x y - 2 4 5 - e t h y l - B - h o m o - 7 - o x a - 5 a cholestan-6-one.


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BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS

t h a t Distylium f a c t o r s m i g h t be b r a s s i n o s t e r o i d s , a n d we t h u s b e g a n to r e i n v e s t i g a t e those plant growth promoting steroids d i s t r i b u t e d i n v a r i o u s p l a n t s o u r c e s u s i n g L J T . A t t h e same time, many J a p a n e s e c h e m i s t s s t a r t e d to s e a r c h f o r n a t u r a l l y o c c u r r i n g brassinosteroids i n plants. After b r a s s i n o s t e r o i d s were identified i n Chinese cabbage s h o o t s ( i 0 , i i ) , followed b y g r e e n t e a l e a v e s ( i £ i 3 ) , we t r i e d to i d e n t i f y Distylium f a c t o r s A a n d B i n t h e f r e s h h e a l t h y l e a v e s of Distylium racemosum , w h i c h w e r e a g a i n c o l l e c t e d i n t h e s u b u r b s of A n j o c i t y i n 1982. (See Figure 1.)


I d e n t i f i c a t i o n o f Distylium F a c t o r s A i a n d B as B r a s s i n o s t e r o i d s T h e e t h y l a c e t a t e - s o l u b l e n e u t r a l f r a c t i o n p a r t i t i o n e d from f r e s h I s u n o k i l e a v e s (16 k g ) w a s p u r i f i e d b y s u c c e s s i v e chromatographic separations on silica gel (/2-hexane-EtOAc-EtOH), A m b e r l i t e X A D - 2 (H20-EtOH), a n d o n s i l i c a g e l f o r p r e p a r a t i v e H P L C (1 x 100cm, C H C h - E t O H ) to g i v e a n a c t i v e f a c t i o n . T h e a c t i v e f r a c t i o n was f u r t h e r p u r i f i e d b y r e p e a t e d H P L C o n F u j i 0 D S - Q 3 (2 x 25cm, CH3CN:H20=60:40) a n d o n D e v e l o s i l ODS-5 (4.6 x 250mm, CH3CN:H2O=50:50) to a f f o r d t h e t w o a c t i v e s u b s t a n c e s . T h e p o l a r fraction appeared to c o n t a i n a s u b s t a n c e chromatographically s i m i l a r to b r a s s i n o s t e r o i d - l a c t o n e itself, while the less polar fraction contained a brassinosteroid-ketone-like substance. Final i d e n t i f i c a t i o n o f t h e s e a c t i v e s u b s t a n c e s was c a r r i e d o u t b y G C - M S a n a l y s i s a f t e r c o n v e r s i o n to t h e b i s m e t h a n e b o r o n a t e derivative, w h i c h was e s t a b l i s h e d b y D r . I k e k a w a a n d h i s c o - w o r k e r s as a microanalytical method for b r a s s i n o s t e r o i d s (14). Four b r a s s i n o s t e r o i d s w e r e i d e n t i f i e d from t h e I s u n o k i l e a v e s , namely b r a s s i n o l i d e a n d n o r b r a s s i n o l i d e from the p o l a r f r a c t i o n corresponding to Distylium factor B, and their biosynthetic precursors, castasterone and brassinone from t h e l e s s polar f r a c t i o n c o r r e s p o n d i n g to Distylium f a c t o r A i . Distylium f a c t o r A2 has y e t to be i d e n t i f i e d , b u t i s p r o b a b l y a 2 - d e o x y - t y p e b r a s s i n o s t e r o i d , w h i c h may be t h e b i o s y n t h e t i c p r e c u r s o r of brassinosteroid-ketone, castasterone a n d / o r brassinone. Thus, the Distylium f a c t o r s w e r e f o u n d to be b r a s s i n o s t e r o i d s ( 7 5 ) . T h e amount of e n d o g e n o u s BS obtained b y the GC/MS method was estimated to be approximately 156ng of n o r b r a s s i n o l i d e , 2 3 n g of b r a s s i n o l i d e , 16ng o f b r a s s i n o n e , 133ng of c a s t a s t e r o n e p e r one k i l o g r a m f r e s h w e i g h t l e a v e s . H o w e v e r , t h i s q u a n t i f i c a t i o n d a t a i s c o n s i d e r e d to be a n u n d e r e s t i m a t e d u e to l o s s e s e n c o u n t e r e d d u r i n g sample p u r i f i c a t i o n , b u t i t s t i l l r e m a i n s q u i t e a small q u a n t i t y .

A Modified Method Brassinosteroids

of

the

Rice

Lamina

Joint

Test

for

T h e L J T b i o a s s a y was d e v e l o p e d f o r a n a u x i n b i o a s s a y b y D r . M a e d a of N a g o y a U n i v e r s i t y i n 1965(i9. A s m e n t i o n e d a b o v e , t h e L J T i s e x t r e m e l y s e n s i t i v e a n d s p e c i f i c t o B S . We e s t a b l i s h e d a


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ABE&MARUMO

23

Research in Japan

OH

OH

H0

V

HO-'^^y

H0'-'^^]|-o 0

0

brassinolide

norbrassinolide


OH

HO

OH

v

HO'' castasterone

Figure leaves.

1.

Brassinosteroids

brassinone

identified

from

Distylium

racemosum

m i c r o - q u a n t i t a t i v e a n a l y t i c a l method f o r BS b i o a s s a y a n d examined t h e s e n s i t i v i t y of v a r i o u s r i c e c u l t i v a r s to d e t e r m i n e suitable e x p e r i m e n t a l c o n d i t i o n s . We s e l e c t e d t h e I t a l i a n c u l t i v a r A r b o r i o as one of t h e most suitable for this brassinosteroid bioassay system(i6). A f t e r s o a k i n g r i c e s e e d s i n a 0.25% a q u e o u s s o l u t i o n of B e n l a t e ( f u n g i c i d e ) f o r 4 8 h r , t h e s e e d s were c u l t i v a t e d at 28-29 °C i n d a r k n e s s on a plastic net floating on distilled water i n a plastic t r a y . L a m i n a j o i n t s e c t i o n s were e x c i s e d from e t i o l a t e d 6 ~ 7 - d a y s e e d l i n g s , w h i c h c o n s i s t e d of t h e s e c o n d leaf l a m i n a ( 0 . 5 - l c m l o n g ) , t h e s e c o n d lamina j o i n t , a n d t h e s e c o n d leaf s h e a t h ( 0 . 5 - l c m l o n g ) . These s e c t i o n s w e r e floated o n d i s t i l l e d w a t e r f o r 2 4 h r . S e c t i o n s , u n i f o r m l y b e n t to a n a n g l e of a b o u t 30 d e g r e e s b e t w e e n t h e l a m i n a a n d t h e s h e a t h were t h e n s e l e c t e d a n d s e v e n to t e n of t h e s e c t i o n s w e r e i n c u b a t e d i n o n e m l o f 2 . 5 mM a q u e o u s p o t a s s i u m maleate b u f f e r s o l u t i o n c o n t a i n i n g t h e t e s t sample. A f t e r i n c u b a t i o n f o r 4 8 h r u n d e r t h e same c o n d i t i o n s , t h e m a g n i t u d e of the angle i n d u c e d between the lamina a n d the sheath was m e a s u r e d . E v e r y o p e r a t i o n was c a r r i e d o u t u n d e r red light w a v e l e n g t h s , e x c e p t f o r t h e f i n a l measurement s t e p of t h e a n g l e . T h i s modified p r o c e d u r e i s now t h e s t a n d a r d method f o r BR detection i n Japan. Conclusion The b e g i n n i n g of b r a s s i n o s t e r o i d r e s e a r c h i n J a p a n was i n i t i a t e d w i t h Distylium f a c t o r r e s e a r c h . Distylium f a c t o r s were d i s c o v e r e d i n Distylium racemosum l e a v e s , w h e n b i o a s s a y e d , u s i n g



24

BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS L J T a n d w e r e f o u n d to be b r a s s i n o s t e r o i d s as e x p e c t e d . The amount of f o u r b r a s s i n o s t e r o i d s was e s t i m a t e d to be l o w e r t h a n a b o u t 150ng i n one k i l o g r a m of f r e s h l e a v e s of Distylium racemosum. The small a m o u n t s o b t a i n e d make i t e a s y to see t h a t t h e q u a n t i t y of c r u d e Distylium f a c t o r s p u r i f i e d i n 1968 h a d b e e n too small to allow e l u c i d a t i o n of the c h e m i c a l s t r u c t u r e . The L J T f o r p u r i f i c a t i o n of Distylium f a c t o r a n d Corn f a c t o r was s h o w n to be the most s u i t a b l e b i o a s s a y method f o r i d e n t i f y i n g naturallyo c c u r r i n g b r a s s i n o s t e r o i d . It i s w e l l - k n o w n t h a t s u c c e s s i v e l y new a n a l o g u e s of b r a s s i n o l i d e w e r e i s o l a t e d a n d i d e n t i f i e d from many k i n d s of p l a n t s u s i n g t h i s b i o a s s a y . B r a s s i n o l i d e i s the first steroid compound with plant growth-promoting a c t i v i t y to be d i s c o v e r e d i n p l a n t s . The u n i q u e c h e m i c a l s t r u c t u r e a n d p l a n t p h y s i o l o g i c a l a c t i v i t y of b r a s s i n o l i d e and its analogues has a t t r a c t e d the s p e c i a l i n t e r e s t s of o r g a n i c c h e m i s t s a n d plant physiologists. Literature Cited

1. Mitchell, J. W.; Mandava, N. ; Worley, J. F.; and Plimmer, J. R. Nature 1970, 225, 1065. 2. Maeda, E. Physiol. Plant 1965, 18, 813. 3. Marumo, S.; Hattori, H.; Abe, H.; Nonoyama, Y. and Munakata, K. Agric. Biol. Chem. 1968, 32, 528. 4. Munakata, K.; Kato, N. and Ikeda, M. "Plant Growth Substances 1973," Hirokawa Publishing Co. Inc., Tokyo, 1974, pp.39-43. 5. Grove, M. D.; Spencer, W. K.; Rohwedder, W. K.; Mandava, N.; Worley, J. F.; Warthen, J. D.; Steffens, G. L.; Flippen-Anderson, J. L. and Cook, J. C. Jr. Nature 1979, 281, 218. 6. Chem. & Eng News 1979, No. 5, 20. 7. Mori, K. Agric. Biol. Chem. 1980, 44, 1211. 8. Ishiguro, M.; Takatsuto, S.; Morisaki, M. and Ikekawa, N. J. Chem. Soc. Chem. Commun. 1980, 962. 9. Wada, K.; Marumo, S.; Ikekawa, N.; Morisaki, M. and Mori, K. Plant Cell Physiol. 1981, 22, 323. 10. Abe, H.; Morishita, T.; Uchiyama, M.; Marumo, S.; Munakata, K.; Takatsuto, S. and Ikekawa, N. Agric. Biol. Chem. 1982, 46, 2609. 11. Abe, H.; Morishita, T.; Uchiyama, M.; Takatsuto, S. and Ikekawa, N. Agric. Biol. Chem. 1984, 48, 2171. 12. Morishita, T.; Abe, H.; Uchiyama, M.; Marumo, S; Takatsuto, S. and Ikekawa, N. Phytochemistry 1983, 22, 1051. 13. Abe, H.; Morishita, T.; Uchiyama, M.; Takatsuto, S.; Ikekawa, N.; Ikeda, M.; Sassa, T.; Kitsuwa, T. and Marumo, S. Experientia 1983, 39, 351. 14. Takatsuto, S.; Ying, B.; Morisaki, M. and Ikekawa, N. J. Chromatogr. 1982, 239, 233. 15. Ikekawa, N.; Takatsuto, S.; Kitsuwa, T.; Saito, H.; Morishita, T. and Abe, H. J. Chromatogr. 1984, 290, 289. 16. Wada, K.; Marumo, S.; Abe, H.; Morishita, T.; Nakamura, K.; Uchiyama, M. and Mori, K. Agric. Biol. Chem. 1984, 48, 719. R E C E I V E D May 8, 1991


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