RiceLamina Inclination, Endogenous Levels in Plant Tissues and

Chapter 17

Downloaded by NORTH CAROLINA STATE UNIV on January 9, 2013 | http://pubs.acs.org Publication Date: November 4, 1991 | doi: 10.1021/bk-1991-0474.ch017

Rice—Lamina Inclination, Endogenous Levels in Plant Tissues and Accumulation during Pollen Development of Brassinosteroids Hiroshi Abe Department of Plant Protection, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan

Search for brassinosteroid [BS] by using the bioassay system of rice-lamina joint test [LJT] demonstrated that BS was present in all parts of plants. BS content in vegetative tissue was extremely low compared with those of known phytohormones, but that in plant male gametophyte (pollen) tissue was exceptionally high. Enrichment of the reproductive tissue with BS, especially typhasterol is enigmatic. Change in the levels of the BS activity was observed during maturation of pollen. Through the measurement of the rate of rice lamina -inclination, a certain difference was observed between brassinolide and IAA.

S i n c e t h e d i s c o v e r y of b r a s s i n o l i d e , t e n y e a r s a g o , r e s e a r c h o n b r a s s i n o l i d e a n d r e l a t e d s t e r o i d s has a t t a i n e d a r e m a r k a b l e d e g r e e of d e v e l o p m e n t . So f a r , twenty-three brassinosteroids [BS] h a v e b e e n i s o l a t e d a n d i d e n t i f i e d from twenty-two plant species. Furthermore, many unknown b r a s s i n o l i d e - l i k e a c t i v e s u b s t a n c e s h a v e been d e t e c t e d , so t h a t t h e n u m b e r of i d e n t i f i e d ones i s l i k e l y to i n c r e a s e i n t h e f u t u r e . T h e b a c k g r o u n d to s u c c e s s f u l i s o l a t i o n of new B S from v a r i o u s p l a n t s o u r c e s i s d u e to t h e u s e of a r i c e - l a m i n a i n c l i n a t i o n t e s t f o r screening that is sensitive and specific for brassinosteroids. In t h i s p a p e r , I w i l l d e s c r i b e : Search for naturally o c c u r r i n g brassinosteroid and endogenous levels i n plants. (2) B r a s s i n o s t e r o i d a c c u m u l a t i o n d u r i n g d e v e l o p m e n t of p l a n t male g a m e t o p h y t e . (3) B r a s s i n o s t e r o i d - i n d u c e d r i c e - l a m i n a i n c l i n a t i o n r e s p o n s e .

(1)

0097-6156/91/0474-0200$06.00/0 © 1991 American Chemical Society In Brassinosteroids; Cutler, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

201

17. A B E Pollen Development of Brassinosteroids


1. 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 B r a s s i n o s t e r o i d a n d Levels in Plants.

Endogenous

We a r e able to r e a d i l y 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 b r a s s i n o s t e r o i d u s i n g r i c e - l a m i n a j o i n t t e s t [ L J T ] (1) as the b i o a s s a y method a n d G C / M S as t h e m i c r o a n a l y t i c a l method, w h i c h was e s t a b l i s h e d by Ikekawa and h i s c o - w o r k e r s as a m i c r o a n a l y t i c a l method f o r B S (2). P l a n t m a t e r i a l was e x t r a c t e d t h r e e times i n methanol o r 80% methanol-H20. A f t e r t h e e x t r a c t was evaporated u n d e r r e d u c e d p r e s s u r e , the r e s u l t i n g aqueous residue was e x t r a c t e d w i t h E t O A c at pH3 a n d t h e E t O A c f r a c t i o n was p a r t i t i o n e d w i t h Na2C03-NaHC03 b u f f e r at pHIO to get neutral substances. The n e u t r a l E t O A c - s o l u b l e s u b s t a n c e s a f t e r e v a p o r a t i o n was t h e n p u r i f i e d o n a small a l u m i n u m oxide ( l O g , M e r c k A l u m i n u m oxide 90, I I - I I I , 70-230mesh) w h i c h was u s e d to b u i l d a small c o l u m n i n a g l a s s s y r i n g e b a r r e l ( i . d . 1.8cm, l e n g t h 3.5cm). T h e sample was d i s s o l v e d i n 10ml of E t O A c - / ? - h e x a n e (4:1, v / v ) a n d l o a d e d onto t h e c o l u m n , w h i c h was w a s h e d w i t h a n a d d i t i o n a l 40ml of t h e same s o l v e n t s y s t e m . A f t e r w a s h i n g , 50ml of E t O H - E t O A c (4:1, v / v ) , w h i c h e l u t e s t h e B S , was p a s s e d u n d e r pressure t h r o u g h t h e c o l u m n . The BS e l u e n t was d i s s o l v e d i n 80% m e t h a n o l H2O (20ml) a n d p a s s e d u n d e r p r e s s u r e at 2 - 3 m l / m i n t h r o u g h t h e first Cis-reversed-phase B o n d E l u t c a r t r i d g e c o l u m n (size 6cc, A n a l y t i c h e m I n t e r n a t i o n a l ) . The c o l u m n t h e n was w a s h e d w i t h a n a d d i t i o n a l 20ml of 80% methanol-H20. A n a d d i t i o n a l 24ml of w a t e r was a d d e d to the c o m b i n e d e l u a t e s of 80% methanol a n d the r e s u l t a n t 50% methanol s o l u t i o n was p a s s e d t h r o u g h s e c o n d C i s Bond Elut column. T h e s e c o n d c o l u m n was t h e n w a s h e d w i t h t h e same s o l v e n t of 20ml. S u b s e q u e n t e l u t i o n of the c o l u m n w i t h 20ml of 100% MeOH e l u t e d BS r e t a i n e d o n t h e c o l u m n a n d was t a k e n to d r y n e s s u n d e r r e d u c e d p r e s s u r e , from w h i c h a l i q u o t s c o u l d be t a k e n f o r t h i n layer chromatography (Kieselgel 0.25mm t h i c k n e s s , M e r c k ) . T h i n - l a y e r c h r o m a t o g r a m , w h i c h was d e v e l o p e d w i t h E t O A c / E t O H (22:3, v / v ) s o l v e n t s y s t e m a n d d e v e l o p m e n t d i s t a n c e s of 15cm, was d i v i d e d into fifteen equal zones a n d after elution with a suitable s o l v e n t , e a c h eluate was t a k e n f o r b i o a s s a y . B S s t a n d a r d s a r e s e p a r a t e d o n t h e T L C d e v e l o p e d w i t h t h e same s o l v e n t s y s t e m a n d the RF v a l u e s a r e : n o r b r a s s i n o l i d e , 0.22; b r a s s i n o l i d e , 0.24; h o m o b r a s s i n o l i d e , 0.27 ( l a c t o n e g r o u p ) ; b r a s s i n o n e , 0.32; c a s t a s t e r o n e , 0.35; e t h y l b r a s s i n o n e , 0.38 ( k e t o n e group); typhasterol, 0.51; t e a s t e r o n e , 0.50 ( 2 - d e o x y k e t o n e g r o u p ) . T h e y a p p e a r i n t h e r a n g e o f RF 0 . 2 t o 0 . 5 a n d i t i s r e a d i l y d i s t i n g u i s h e d b y t h e RF v a l u e s b e t w e e n t h e l a c t o n e g r o u p a n d t h e ketone group, and also between the ketone g r o u p and the 2 d e o x y k e t o n e g r o u p . T h u s , T L C s h o u l d be f a v o r a b l e f o r m a k i n g p r e l i m i n a r y e s t i m a t i o n of e n d o g e n o u s B S i n p l a n t s . H o w e v e r , T L C i s no l o n g e r d i s t i n g u i s h a b l e from B S w i t h t h e s t r u c t u r a l v a r i a t i o n w i t h i n t h e same g r o u p . The p r o b l e m c o u l d be s o l v e d b y the use of H P L C . The c r u d e BS f r a c t i o n a f t e r p u r i f i c a t i o n o n t h e B o n d E l u t c o l u m n was t h e n c h r o m a t o g r a p h e d o n a r e v e r s e d - p h a s e Ci8 D e v e l o s i l H P L C c o l u m n ( i . d . 4.6mm x 25cm) e l u t i n g w i t h CH3CN-H2O

6OF254,

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


202

BRASSINOSTEROIDS: CHEMISTRY, BIOACTIVITY, AND APPLICATIONS

(75:25, v / v ) o r CH3CN-H2O (45:55, v / v ) . E a c h f r a c t i o n was t h e n t a k e n to d r y n e s s , from w h i c h a n a l i q u o t was b i o a s s a y e d b y L J T . The a c t i v e f r a c t i o n s were c o m b i n e d a n d a n a l y z e d b y G C / M S as t h e bismethaneboronate a n d / o r methaneboronate trimethylsilyl ether derivatives. The L J T b i o a s s a y , c o u p l e d w i t h the m i c r o a n a l y t i c a l t e c h n i q u e s u s i n g G C / M S , l e d to the i d e n t i f i c a t i o n of new c o n g e n e r s of b r a s s i n o s t e r o i d w i t h s t r u c t u r a l v a r i a t i o n s from d i v e r s e p l a n t s p e c i e s a n d p l a n t t i s s u e s , s u c h as some s t e r e o i s o m e r s of h y d r o x y l a n d a l k y l - g r o u p s i n the A - r i n g a n d s i d e c h a i n , as s h o w n i n T a b l e I. The amount was l e s s t h a n one m i c r o g r a m p e r k i l o g r a m fresh weight, i n d i c a t i n g that the amount was quite small. F u r t h e r m o r e , many u n k n o w n B S - l i k e a c t i v e s u b s t a n c e s h a v e b e e n d e t e c t e d , so t h a t t h e n u m b e r of i d e n t i f i e d B S i s l i k e l y to i n c r e a s e i n t h e f u t u r e . T h u s , s e a r c h f o r B S has d e m o n s t r a t e d t h a t B S i s p r e s e n t i n a l l p a r t s of v e g e t a t i v e t i s s u e s of p l a n t s , t h a t t h e a m o u n t s i n t h e t i s s u e s were l e s s t h a n one fig i n one k i l o g r a m f r e s h w e i g h t , so i t was e x t r e m e l y low as c o m p a r e d w i t h t h o s e of k n o w n p h y t o h o r m o n e s , a n d a l s o t h a t e n d o g e n o u s BS d i f f e r s among plant species and plant tissues.

2. B r a s s i n o s t e r o i d A c c u m u l a t i o n d u r i n g D e v e l o p m e n t a n d G r o w t h of P l a n t Male G a m e t o p h y t e . P o l l e n i s s h o w n to be a r i c h s o u r c e of p l a n t g r o w t h s u b s t a n c e s . When b i o a s s a y e d b y the L J T , a n e x t r a c t from the p l a n t male g a m e t o p h y t e ( p o l l e n ) w e r e f o u n d to c o n t a i n a n e x c e p t i o n a l l y h i g h c o n t e n t of B S . The 80% a c e t o n i t r i l e - w a t e r s o l u b l e f r a c t i o n o b t a i n e d from a m e t h a n o l - c h l o r o f o r m e x t r a c t of Lilium longiflorum p o l l e n was w a s h e d w i t h i?-hexane, a n d s e p a r a t e d into its neutral and acidic fractions. As d e s c r i b e d above, the n e u t r a l f r a c t i o n was p u r i f i e d on a n a l u m i n u m a d s o r p t i o n c o l u m n a n d Ci8 B o n d E l u t c a r t r i d g e c o l u m n . The a c t i v e f r a c t i o n was subjected to reversed-phase Ci8 Develosil HPLC, using the a c e t o n i t r i l e - a q u e o u s mobile p h a s e . B y H P L C , t h r e e f r a c t i o n s w i t h quite h i g h biological a c t i v i t y were s e p a r a t e d . E a c h f r a c t i o n was analyzed b y GC/MS after c o n v e r t i n g to the methaneboronate and/or methaneboronate trimethylsilyl ether by the method mentioned above. The main BS found in l i l y pollen were typhasterol (2-deoxycastasterone), castasterone and brassinolide. We a l s o i d e n t i f i e d e n d o g e n o u s BS from t h e p o l l e n of s e v e r a l k i n d s of p l a n t s , t h a t i s , t y p h a s t e r o l , teasterone, castasterone and b r a s s i n o l i d e i n Citrus unshiu ( o r a n g e ) ; t y p h a s t e r o l a n d t e a s t e r o n e in Typha latifolia (cat t a i l ) ; t y p h a s t e r o l i n Tulipa gesneriana ( t u l i p ) ; c a s t a s t e r o n e i n Thea sinensis ( g r e e n tea). The amount of typhasterol, which i s a main BS i n p o l l e n of o r a n g e , t u l i p , l i l y , c a t t a i l , was one h u n d r e d i± g o r more i n one k i l o g r a m f r e s h w e i g h t of l i l y pollen. Thus, the plant reproductive tissue, e s p e c i a l l y p l a n t p o l l e n , was c o n f i r m e d to be one of the r i c h e s t s o u r c e s of BS c o m p a r e d w i t h t h a t of p l a n t v e g e t a t i v e t i s s u e . T h i s f a c t s u g g e s t s t h a t B S p o s s e s s e s a p h y s i o l o g i c a l f u n c t i o n i n the


Pollen Development of Brassinosteroids

ABE

Table I .


Brassino steroid I II ID IV V VI vn VI K

Amounts o f B r a s s i n o s t e r o i d s Isunoki (ng/kg) 156 23

-16 133

-

Green t e a (ng/kg) _

4.6

-

2.0 110.0 trace

-

id. id.

i n Higher

Cabbage (ng/kg) 1.3 9.4 trace 0.78 1.6 0.13

-

203 Plants

Rice plant (ng/kg)

-

-

trace

id.

13.6

id.

-

-

8.4

i d . : i d e n t i f i e d but not q u a n t i f i e d . I s u n o k i : Leaves (3); Green t e a : Leaves (4) Cabbage : Immature seeds and s h e a t h s (5) R i c e p l a n t : Shoots (ff) Q u a n t i f i c a t i o n d a t a p r e s e n t e d a r e u n d e r e s t i m a t e s due l o s s e s encountered d u r i n g sample p u r i f i c a t i o n .

OH R

Mung bean (ng/kg)

Norbrassinolide

to

(R=H, I )

B r a s s i n o l i d e (R=CH3, II) H o m o b r a s s i n o l i d e (R=CH2CH3, ID)

Brassinone

(R=H, IV)

C a s t a s t e r o n e (R=CH3, V ) Ethylbrassinone Dolichosterone

Typhasterol

(R=CH2CH3, VI) (R=CH2, VII)

( R = a - 0 H , VI)

T e a s t e r o n e ( R = £ - 0 H , IX)



204


r e g u l a t i o n of r e p r o d u c t i v e g r o w t h a n d also p o l l e n i s a v e r y u s e f u l m a t e r i a l f o r s t u d y i n g the b i o s y n t h e s i s of B S . Liliaceous plant exhibit a close relationship between the f l o w e r b u d l e n g t h a n d t h e d e v e l o p m e n t a l s t a g e of p o l l e n (7). P o l l e n of Lilium longiflorum c v . G e o r g i a was g r o u p e d a n d c o l l e c t e d i n t o s i x c l a s s e s , among d e v e l o p m e n t a l s t a g e s from a n immature u n i n u c l e a t e p o l l e n a f t e r meiosis, to a mature b i n u c l e a t e p o l l e n a f t e r a n t h e s i s , b a s e d on t h e f l o w e r b u d l e n g t h . U s i n g 500 of t h e male r e g e n e r a t i v e t i s s u e s from e a c h s t a g e , the BS f r a c t i o n was s e p a r a t e d a n d p u r i f i e d b y a simple a n d r a p i d method, w h i c h consists of 5 s t e p s , namely, methanol-chloroform extraction, acetonitrile-hexane partition, ethyl acetate-buffer p a r t i t i o n (in o r d e r to s e p a r a t e neutral substances only), alumina column chromatography and Ci8 Bond Elut cartridge column c h r o m a t o g r a p h y . A n a l i q u o t , e q u i v a l e n t to 0.5 male t i s s u e s , was t a k e n f o r the L J T . Stage V, j u s t before anthesis, had the g r e a t e s t BS a c t i v i t y ( F i g u r e 1). The same c h a n g e i n t h e l e v e l s of BS a c t i v i t y was o b s e r v e d d u r i n g the d e v e l o p m e n t of g r e e n t e a (Thea sinensis) p o l l e n . T h e a c t i v e f r a c t i o n of S t a g e s I, V a n d V I was f u r t h e r p u r i f i e d b y r e v e r s e d - p h a s e Cis D e v e l o s i l H P L C , to d i s t i n g u i s h between two a c t i v i t i e s by t y p h a s t e r o l and by c a s t a s t e r o n e a n d / o r b r a s s i n o l i d e . A n a l i q u o t , e q u i v a l e n t to 10 male t i s s u e s , was a p p l i e d to t h e b i o a s s a y . BS a c t i v i t y i n the Stage I was d u e to a t r a c e amount of t y p h a s t e r o l o n l y , a n d t h a t n o t e d i n S t a g e s V a n d V I , c o m p o s e d of the c o m b i n e d a c t i v i t y of t y p h a s t e r o l a n d c a s t a s t e r o n e / b r a s s i n o l i d e . The a c t i v i t y of b o t h f r a c t i o n s was almost t h e same. A s the r e l a t i v e a c t i v i t y of t y p h a s t e r o l i s one t e n t h l o w e r t h a n t h a t of c a s t a s t e r o n e , a n d one t w e n t i e t h t h a t of b r a s s i n o l i d e i n t h i s b i o a s s a y s y s t e m , t y p h a s t e r o l was d e t e c t e d at a v e r y h i g h concentration i n the endogenous levels. The respective amounts of BS i n t h e s t a g e s I, V a n d V I were e s t i m a t e d , b y b i o a s s a y , to be a p p r o x i m a t e l y 22, 235 a n d 186 / z g , r e s p e c t i v e l y , i n one k i l o g r a m f r e s h w e i g h t of t h e male r e p r o d u c t i v e t i s s u e as s h o w n i n T a b l e II. We also a n a l y z e d p h y t o s t e r o l i n t h e male t i s s u e s of S t a g e s I a n d V I . The main s t e r o l s i n b o t h s t a g e s w e r e £ - s i t o s t e r o l a n d c a m p e s t e r o l , b u t the r a t i o of c a m p e s t e r o l to j3s i t o s t e r o l was a b o u t 1:1 at Stage I, a n d was a b o u t 1:3 at Stage V I . The e n d o g e n o u s BS were f o u n d to c h a n g e i n the l e v e l s of q u a n t i t y a n d q u a l i t y d u r i n g m a t u r a t i o n of p o l l e n . E n r i c h m e n t of t h e l i l y ' s r e p r o d u c t i v e tissue with t y p h a s t e r o l is enigmatic but is useful for e s t a b l i s h i n g t h e b i o s y n t h e t i c p a t h w a y s a n d metabolic fates of t y p h a s t e r o l , c a s t a s t e r o n e a n d b r a s s i n o l i d e i n p l a n t s a n d also t h e relationship between endogenous BS levels and t h e i r p h y s i o l o g i c a l significance in plant growth regulation, especially i n reproductive growth regulation.

3. B r a s s i n o s t e r o i d - i n d u c e d R i c e - l a m i n a I n c l i n a t i o n R e s p o n s e A rice-lamina joint is a v e r y important tissue i n regulating t h e i n c l i n a t i o n of t h e leaf b l a d e . B r a s s i n o l i d e was t e n t h o u s a n d times h i g h e r i n a c t i v i t y o n l a m i n a i n c l i n a t i o n t h a n I A A . The r e a s o n



17. ABE


205

8 160°J u Q BR '8xl0- M

60

140°

I

9

120°-

Cont.

CO

PQ

i

ii

in

iv

v

vi

24 I 40

40 I 58

58 I 70

70 I 120

120 I 170

-g

I

Developmental Stage of Pollen (Flower Bud Length, mm) Figure 1. Change in the level of brassinosteroid activity during the development of pollen of L. longiflorwn c.v. Georgia. Equivalent to 0.5 male tissues was taken for the LJT. BR: brassinolide.


206


T a b l e I I . Amount o f B r a s s i n o s t e r o i d i n T i s s u e o f Lilium longiflorum c v . G e o r g i a Developmental stage of p o l l e n


I V VI

Male

Reproductive

B r a s s i n o s t e r o i d Amount Typhasterol Ug/kg)

Stage Stage Stage

the

22 210 180

Castasterone•Brassinolide (//g/kg) N.D 25 6

S t a g e s I,V a n d VI show d e v e l o p m e n t a l s t a g e s of a n immature u n i n u c l e a t e p o l l e n j u s t a f t e r t h e meiosis, a mature binucleate p o l l e n j u s t before a n d a f t e r the a n t h e s i s , r e s p e c t i v e l y . F r e s h w e i g h t of e a c h s t a g e is 85.0, 178.4, a n d 95.5 mg i n one male t i s s u e , r e s p e c t i v e l y . T h e B S amount was e s t i m a t e d from t h e b i o l o g i c a l a c t i v i t y i n L J T of e a c h a c t i v e f r a c t i o n a f t e r H P L C .

f o r t h e h i g h s p e c i f i c r e s p o n s e of r i c e - l a m i n a j o i n t c e l l s to BS i s s t i l l o b s c u r e . A r i c e - l a m i n a j o i n t s e c t i o n i s composed of 2 n d leaf b l a d e , 2 n d leaf s h e a t h , 3 r d leaf, lamina j o i n t , a u r i c l e (meaning leaf e a r ) , a n d l i g u l e (meaning leaf t o n g u e ) . The i n c l i n a t i o n at the l a m i n a j o i n t has b e e n k n o w n to r e s u l t from more e x p a n s i o n of t h e a d a x i a l c e l l s t h a n t h a t of the d o r s a l c e l l s . On t h e o t h e r h a n d , a r i c e c u l t i v a r w i t h o u t the l i g u l e , named M u y o z e t s u i n J a p a n e s e , d i d not r e s p o n d to b r a s s i n o l i d e at the h i g h c o n c e n t r a t i o n of 10~ M, so, t h e l i g u l e , b e i n g i n c o n t a c t w i t h the a d a x i a l c e l l s , was f o u n d to be a p h y s i o l o g i c a l l y i m p o r t a n t t i s s u e i n the l a m i n a i n c l i n a t i o n . I n o r d e r to s e l e c t a s u i t a b l e r i c e c u l t i v a r f o r m i c r o - q u a n t i t a t i v e b i o a s s a y , we examined t h e s e n s i t i v i t y of a b o u t s i x t y t y p e s of r i c e cultivars. E i g h t e e n c u l t i v a r s were judged sensitive enough. A r b o r i o ( I t a l i a n c u l t i v a r ) , Kinmaze a n d N i h o n b a r e ( J a p o n i c a ones) were s e n s i t i v e to BS a n d u s e f u l c u l t i v a r s . A r b o r i o , e s p e c i a l l y , g r e w the t a l l e s t , a b o u t 14cm, a n d i t was e a s y to c u t s e g m e n t s from t h e s e s e e d l i n g s . I n g e n e r a l , we h a v e u s e d the Arborio cultivar in s c r e e n i n g t e s t s of n a t u r a l l y - o c c u r r i n g BS a n d i n m i c r o - q u a n t i t a t i v e b i o a s s a y of B S . The c o r r e l a t i o n b e t w e e n the i n d u c e d a n g l e s of the l a m i n a j o i n t a n d the c o n c e n t r a t i o n of b r a s s i n o l i d e a n d c a s t a s t e r o n e was d e t e r m i n e d u s i n g A r b o r i o a n d N i h o n b a r e ; a l i n e a r c o r r e l a t i o n was o b t a i n e d b e t w e e n 5 x 10" and 5 x IO / z g / m l . When m e a s u r e d e v e r y 90 m i n u t e s , a n g l e s of l a m i n a joints induced by 6-ketone-type BS, that is, brassinone, castasterone and ethylbrassinone, and their corresponding lactones, norbrassinolide, brassinolide and homobrassinolide, increased l i n e a r l y d u r i n g the initial 24hr incubation, then g r a d u a l l y l e v e l e d off. T h e 6 - k e t o n e c o m p o u n d s s h o w e d no time l a p s e (lag p h a s e ) f o r t h e i r a c t i v i t y , w h e n c o m p a r e d w i t h t h e i r lactones. These r e s u l t s s u g g e s t that these 6-ketone compounds t h e m s e l v e s affect t h e l a m i n a j o i n t s w i t h o u t b e i n g c o n v e r t e d to t h e i r r e s p e c t i v e l a c t o n e s (5). 7

8

- 5


17. ABE


207


T h r o u g h t h e measurement of t h e r a t e of l a m i n a - i n c l i n a t i o n , it is possible to observe a c e r t a i n difference between brassinolide a n d I A A . A n e x c i s e d l a m i n a - j o i n t segment w i t h t h e whole of t h e 2 n d leaf b l a d e was u s e d i n t h i s e x p e r i m e n t . T h e segment w a s floated o n d i s t i l l e d w a t e r f o r 18 h o u r s a f t e r c u t t i n g , a n d a f t e r was transferred t o p o t a s s i u m maleate buffer solution, then p r e i n c u b a t e d f o r 2 h o u r s , i t was t h e n c h a r g e d t o t h e t e s t s o l u t i o n of BS a n d I A A f o r 4 t o 5 h o u r s u n d e r t h e c o n d i t i o n s d e s c r i b e d a b o v e . T h e d e g r e e a n g l e w a s m e a s u r e d a t 15 minute i n t e r v a l s . T h e r a t e of l a m i n a i n c l i n a t i o n was d e t e r m i n e d b y the following equation; t h e r a t e = ( A n i n c r e a s e d d e g r e e a n g l e f o r 15 min. i n t e s t s o l u t i o n ) -r ( A n i n c r e a s e d d e g r e e a n g l e f o r 15 m i n . i n b u f f e r s o l u t i o n f o r 2 h o u r s ) x 100. The r a t e of l a m i n a i n c l i n a t i o n i n d u c e d b y B S e x h i b i t s i t s maximum v a l u e a f t e r 120-180 m i n u t e s from t h e b e g i n n i n g of t h e t r e a t m e n t . T h e maximum r a t e i n d u c e d b y I A A i n c o m p a r i s o n t o t h a t b y b r a s s i n o l i d e o c c u r s w i t h i n 30 m i n u t e s a f t e r t r e a t m e n t b e g i n s . We must f i n d o u t t h e p h y s i o l o g i c a l r e a s o n f o r e n c o u n t e r i n g s u c h difference i n the rate of the lamina inclination between brassinolide a n d IAA. Conclusion 1. B S were p r e s e n t i n a l l p a r t s o f v e g e t a t i v e a n d r e p r o d u c t i v e t i s s u e s a t a e x t r e m e l y low c o n c e n t r a t i o n , a n d e n d o g e n o u s B S d i f f e r e d from p l a n t s p e c i e s a n d p l a n t t i s s u e s . 2. P l a n t m a t u r e - p o l l e n w a s r i c h i n t h e B S l e v e l a n d endogenous BS w a s a c t i v e l y p r o d u c e d a n d a c c u m u l a t e d i n p o l l e n d u r i n g t h e g r o w t h a n d d e v e l o p m e n t . T h e r o l e of b r a s s i n o s t e r o i d i n p o l l e n i s u n k n o w n , b u t may be i m p o r t a n t i n f e r t i l i z a t i o n . 3. R i c e - l a m i n a j o i n t i n c l i n a t i o n r e s p o n s e was h i g h l y s e n s i t i v e a n d s p e c i f i c f o r B S . T h e e x p a n s i o n of a d a x i a l c e l l s a t l a m i n a j o i n t s i s c o n s i d e r e d t o be s i g n i f i c a n t p h y s i o l o g i c a l f u n c t i o n s of B S , b u t t h e mechanism i s s t i l l o b s c u r e . Literature Cited 1. Maeda, E. Physiol. Plant 1965, 18, 813. 2. T a k a t s u t o , S.; Ying, B . ; Morisaki, M . ; I k e k a w a , N . J. Chromatogr. 1982, 239, 233. 3. Ikekawa, N.; Takatsuto, S.; Kitsuwa, T.; Saito, H.; Morishita, T.; Abe, H. J. Chromatogr. 1984, 290, 289. 4. Abe, H.; Morishita, T.; Uchiyama, M.; Takatsuto, S.; Ikekawa, N. Agric. Biol. Chem. 1984, 48, 2171. 5. Abe, H.; Morishita, T.; Uchiyama, M.; Marumo, S.; Munakata, K.; Takatsuto, S.; Ikekawa, N. Agric. Biol. Chem. 1982, 46, 2609. 6. Abe, H.; Nakamura, K.; Morishita, T.; Uchiyama, M . ; Takatsuto, S.; Ikekawa, N. Agric. Biol. Chem. 1984, 48, 1103. 7. Tanaka, I.; Ito, M. Plant Sci. Lett. 1980, 17, 729. 8. Wada, K.; Marumo, S.; Abe, H.; Morishita, T.; Nakamura, K.; Uchiyama, M.; Mori, K. Agric. Biol. Chem. 1984, 43, 719. R E C E I V E D May 1, 1991


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