Comparative Studies on the Cell Wall Polymers Obtained from

Chapter 24

Comparative Studies on the Cell Wall Polymers Obtained from Different Parts of Rice Grains

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 12, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch024

Naoto Shibuya National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba, Ibaraki 305, Japan

Cell wall polymers from different parts of rice grain dif fered in both composition and detailed structure. Hemi cellulosic polysaccharides of the cell wall preparations obtained from both outer and inner parts of the grain contained arabinoxylan and xyloglucan. The amount of β-1,3-,1,4-glucan was negligible in the cell walls of the outer part of the grain when compared to that in the en dosperm cell wall. The degree of lignification also differed from tissue to tissue. In addition, the arabinoxylan ob tained from the outer part of the grain carried side-chains with more complicated structures than endospermic ara binoxylan. Similar differentiation on the molecular level is also suggested for other cell wall polymers. In order t o u n d e r s t a n d the process o f differentiation a n d t h e properties o f the r e s u l t i n g cell walls, i t is necessary t o identify the changes t h a t o c c u r i n cell w a l l polymers. Several recent papers suggest t h a t changes i n cell surface carbohydrates i n a n i m a l s a n d p l a n t s m a y be b o t h a cause a n d effect o f differentiation (1-3). O n e possible approach t o the s t u d y o f differentiation is t o c o m p a r e the c o m p o s i t i o n a n d c h e m i c a l s t r u c t u r e o f c e l l - w a l l p o l y m e r s d u r i n g development. C e r e a l grains, such as rice a n d w h e a t , are c o m p o s e d of several tissues w h i c h appear a t different stages o f differentiation. T h u s , s t a r c h e n d o s p e r m originates f r o m fertilized p o l a r n u c l e i , w h i l e the t h i n cell walls are considered t o be p r i m a r y cell walls (4,5). T h e outer p a r t o f the e n d o s p e r m is covered w i t h several layers o f aleuron cells, w h i c h are also derived f r o m the same fertilized p o l a r nucleus b u t w h i c h differentiate i n t o a specific tissue d u r i n g t h e m a t u r a t i o n process. C e l l walls o f the a l e u r o n cells are m u c h thicker t h a n those o f the e n d o s p e r m , a n d are considered t o be secondary walls. T h e o u t e r m o s t p a r t o f the g r a i n is a caryopsis coat consisting o f several compressed cell layers w i t h very t h i c k ( > 1 u m ) w a l l s . 0097-6156/89/0399-0333$06.00/0 Ο 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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G e r m , or e m b r y o , is derived f r o m the f e r t i l i z e d egg a n d consists o f several different tissues. In view of the p r a c t i c a l i m p o r t a n c e of these g r a i n s , for e x a m p l e i n d i e t a r y fibers, i t is useful to s t u d y the cell w a l l p o l y m e r s of their tissues d u r i n g d i f f e r e n t i a t i o n . I n the present w o r k , we used rice grains ( b r o w n rice w i t h o u t husk) as a s t a r t i n g m a t e r i a l . T h e y were f r a c t i o n a t e d i n t o several d i s t i n c t h i s t o l o g i c a l components, a n d the c o m p o s i t i o n a n d d e t a i l e d s t r u c t u r e of cell w a l l p o l y m e r s was c o m p a r e d . Materials and Methods Isolation of Cell Walls from Different Parts of Rice Grain. Rice grains were g r a d u a l l y peeled u s i n g a c o n v e n t i o n a l m i l l i n g i n s t r u m e n t (Satake M o tor O n e - P a s s T y p e Test M i l l , S a t a k e E n g i n e e r i n g C o . , L t d . , T o k y o ) , a n d the fractions r i c h i n specific h i s t o l o g i c a l c o m p o n e n t s were collected. T h e p o t a s s i u m content of these fractions was used as a n i n d e x to identify the f r a c t i o n enriched i n aleuron tissue (6). T h e caryopsis coat f r a c t i o n was o b t a i n e d f r o m the v e r y first m i l l i n g f r a c t i o n . S t a r c h y e n d o s p e r m was o b t a i n e d b y t h o r o u g h l y r e m o v i n g the outer p a r t o f the g r a i n . T h e g e r m f r a c t i o n was separated f r o m the b r a n f r a c t i o n b y s i e v i n g w i t h 10-20 mesh sieves. C e l l walls were o b t a i n e d by the successive e x t r a c t i o n of the defatted tissues w i t h S D S - m e r c a p t o e t h a n o l a n d d i m e t h y l s u l f o x i d e (7). A n e n z y m a t i c m e t h o d usi n g a c o m b i n a t i o n of protease a n d amylase (8) was also used to o b t a i n cell walls f r o m these tissues, a n d the a n a l y s i s of the cell w a l l p r e p a r a t i o n s so o b t a i n e d showed t h a t b o t h methods gave b a s i c a l l y the same p r e p a r a t i o n s . T h e y i e l d of the cell walls ( w / w of defatted tissue) was 0 . 3 % for the e n d o s p e r m , 1 2 % for the g e r m , 2 0 % for the aleuron tissue, a n d 2 9 % for the caryopsis coat. S c a n n i n g electron m i c r o g r a p h s of these cell w a l l p r e p a r a tions ( F i g . 1) showed t h a t b o t h of the cell w a l l p r e p a r a t i o n s o b t a i n e d f r o m the caryopsis coat, a n d the aleuron tissue, were very t h i c k a n d a p p e a r e d of h a r d t e x t u r e . O n the other h a n d , the e n d o s p e r m cell w a l l p r e p a r a t i o n was very t h i n a n d appeared to be of softer t e x t u r e . T h e m a i n p a r t of the cell w a l l p r e p a r a t i o n f r o m the g e r m looked s i m i l a r t o the e n d o s p e r m cell w a l l b u t was somewhat thicker a n d harder. These p r e p a r a t i o n s were free of other c e l l u l a r particles such as s t a r c h or proteins. Results and Discussion Comparison of the Overall Composition of Cell Wall Preparations. A s can be seen f r o m T a b l e I, cell w a l l p r e p a r a t i o n s showed a significant difference i n t h e i r p o l y m e r c o m p o s i t i o n . T h e e n d o s p e r m cell walls resembled p r i m a r y walls, since they were v i r t u a l l y free of l i g n i n b u t r i c h i n pectic substances. O n the other h a n d , the cell w a l l p r e p a r a t i o n s o b t a i n e d f r o m the caryopsis coat a n d the aleuron tissue were h i g h l y lignified, a n d their pectic content was very low. T h e g e r m cell w a l l showed a somewhat i n t e r m e d i a t e c o m p o s i t i o n between these two types, p r o b a b l y reflecting the fact t h a t i t consists of several different tissues. T h e monosaccharide c o m p o s i t i o n of the hemicelluloses, w h i c h were a m a j o r f r a c t i o n of a l l of these cell w a l l p r e p a r a t i o n s , also showed significant differences, especially i n the a m o u n t s of glucose a n d galactose ( T a b l e II).


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335


24.

F i g u r e 1. S c a n n i n g electron m i c r o g r a p h of the cell w a l l p r e p a r a t i o n s ob t a i n e d f r o m the different parts of rice g r a i n (7). C a r y o p s i s coat (upper left), a l e u r o n layer (upper r i g h t ) , germ (lower left) a n d s t a r c h y e n d o s p e r m (lower r i g h t ) . B a r s i n the p i c t u r e i n d i c a t e 5 μπι.


336


T a b l e I. C o m p o s i t i o n of the C e l l W a l l P r e p a r a t i o n s O b t a i n e d f r o m Differ ent H i s t o l o g i c a l F r a c t i o n s


Fraction

Pectic Substances

Hemicellulose

α-Cellulose

Lignin

7 11 27 23

38 42 49 47

28 31 23 21

27 16 1 9

C a r y o p s i s coat A l e u r o n tissue Endosperm Germ

W h i l e the hemicelluloses o b t a i n e d f r o m the g e r m , a l e u r o n , a n d caryopsis coat cell walls a l l showed a s i m i l a r monosaccharide c o m p o s i t i o n , t h i s was not the case for the e n d o s p e r m tissue. T h u s , a m a j o r difference i n the s t r u c t u r e of hemicellulosic polysaccharides exists between the p r e p a r a t i o n s o b t a i n e d f r o m the e n d o s p e r m cell walls a n d those f r o m the cell walls of the other parts of the g r a i n , i.e., rice b r a n . ( R i c e b r a n consists of the c a r y o p sis coat, aleuron layer a n d germ.) C o m p a r i s o n of the detailed s t r u c t u r a l features of the hemicellulosic polysaccharides of e n d o s p e r m a n d b r a n cell walls w i l l be discussed i n the following sections. T a b l e II. M o n o s a c c h a r i d e C o m p o s i t i o n of P e c t i c Polysaccharides a n d H e m i cellulose O b t a i n e d f r o m Different C e l l W a l l P r e p a r a t i o n s N e u t r a l Sugar C o m p o s i t i o n ( m o l %)

Fraction

Rhamnose

Fucose

Arabinose

Xyl ose

Hemicellulose: C a r y o p s i s coat A l e u r o n layer Germ Endosperm

1.0 1.0 1.2 0.9

0.4 0.4 0.5 0.5

35.6 36.7 36.7 26.4

43.6 43.5 38.1 41.1

1.2 1.1 0.7 0.6

43.6 48.9 46.9 33.0

26.8 27.5 20.5 30.4

Pectic Polysaccharides: C a r y o p s i s coat 5.0 A l e u r o n layer 3.6 Germ 2.3 Endosperm 6.1

Uronic

Glu cose

Content (wt.%)

7.4 7.4 8.8 1.9

12.0 11.1 14.7 29.1

13.5 12.8 13.8 12.1

11.9 11.1 10.7 11.4

11.3 7.7 19.0 18.5

31.5 24.9 16.4 34.5

Gal actose

T h e sugar c o m p o s i t i o n of the pectic polysaccharides o b t a i n e d f r o m these cell w a l l preparations (Table II) also suggested differences i n the s t r u c t u r a l features of the different cell w a l l p r e p a r a t i o n s . D e t a i l e d s t r u c t u r a l i n f o r m a t i o n is o n l y available for the pectic polysaccharides o b t a i n e d f r o m the e n d o s p e r m cell w a l l : T h e m a i n f r a c t i o n of e n d o s p e r m pectic p o l y s a c charide was separated i n t o two fractions, a n e u t r a l s u g a r - r i c h f r a c t i o n a n d a f r a c t i o n w i t h a very h i g h content of D - g a l a c t u r o n i c a c i d (8). S t r u c t u r a l



24.

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Cell Wall Polymersfrom Different Parts of Rice

337

analysis o f the former f r a c t i o n i n d i c a t e d a s t r u c t u r e s i m i l a r t o the so-called r h a m n o g a l a c t u r o n a n - I s t r u c t u r e (9), w h i c h consists of a b a c k b o n e o f 1,4l i n k e d g a l a c t u r o n a n chain i n t e r r u p t e d b y 1,2-linked L - r h a m n o s y l residues a n d side-chains r i c h i n 1,5-linked L - a r a b i n o f u r a n o s y l a n d 1,4-linked D g a l a c t o s y l residues (10). T h e y i e l d o f the pectic p o l y s a c c h a r i d e o f the e n d o s p e r m cell w a l l a n d the g e r m cell w a l l is s i m i l a r , b u t the sugar c o m p o s i t i o n is very different, as s h o w n i n T a b l e II. T h e u r o n i c a c i d content is m u c h higher i n the e n d o s p e r m p r e p a r a t i o n . O n the other h a n d , the a m o u n t of arabinose a n d rhamnose is m u c h higher i n the g e r m p e c t i n . N o t e t h a t these p r e p a r a t i o n s are crude fractions a n d further s t r u c t u r a l studies o n the purified fractions are necessary to reach any definitive conclusions. N e v e r theless, these results suggest possible differences i n their s t r u c t u r a l features, such as a difference i n the r a t i o of the n e u t r a l s u g a r - r i c h d o m a i n s a n d the g a l a c t u r o n a n r i c h d o m a i n s , l e n g t h a n d s t r u c t u r e s of side-chains, etc. M e t h y l a t i o n analysis of whole cell w a l l p r e p a r a t i o n (7) also suggested differences i n the c o m p o s i t i o n a n d the s t r u c t u r e o f the c o m p o n e n t p o l y s a c charides. F o r e x a m p l e , most of the arabinose residues i n the e n d o s p e r m cell w a l l were n o n - r e d u c i n g . O n the other h a n d , a large p a r t o f the a r a b i nose residues i n the g e r m cell w a l l were located i n the inner c h a i n p o r t i o n w i t h various types of linkages. S i m i l a r results were also o b t a i n e d i n the case o f the a l e u r o n cell w a l l . T h e 1,3-linked glucose residues were detected p r a c t i c a l l y o n l y i n the e n d o s p e r m cell w a l l p r e p a r a t i o n . T h e s e results s u g gest a significant difference i n the c o m p o s i t i o n a n d d e t a i l e d s t r u c t u r e o f the c o m p o n e n t polysaccharides of these p r e p a r a t i o n s . Structure of the Hemicellulose Polysaccharides from Endosperm and Bran Cell Wall. A s suggested above, there is a m a j o r difference i n the s t r u c t u r e of h e m i c e l l u l o s i c polysaccharides o b t a i n e d f r o m the e n d o s p e r m a n d f r o m the other parts of the g r a i n . T h e hemicellulose p r e p a r a t i o n s f r o m b o t h the e n d o s p e r m a n d b r a n cell walls were further f r a c t i o n a t e d a n d s u b j e c t e d to s t r u c t u r a l analysis u s i n g c h e m i c a l a n d e n z y m a t i c m e t h o d s . F i g u r e 2 s u m m a r i z e s the s t r u c t u r e of the hemicellulosic polysaccharides o b t a i n e d f r o m the e n d o s p e r m cell w a l l (11,12). O v e r t w o - t h i r d s o f the hemicellulose is c o m p o s e d of a r a b i n o x y l a n s , especially a c i d i c a r a b i n o x y l a n s . T h e y are h i g h l y b r a n c h e d , c a r r y i n g m o s t l y very short side chains o f a single a r a b i n o f u r a n o s y l or ( 4 - 0 - m e t h y l ) - g l u c u r o n o s y l residue. T h e e n d o s p e r m h e m i cellulose also c o n t a i n e d two other polysaccharides as m i n o r c o m p o n e n t s , n a m e l y , x y l o g l u c a n a n d / ? - l , 3 - , l , 4 - g l u c a n . B o t h of these c o m p o n e n t s were firmly associated w i t h a s m a l l a m o u n t of a r a b i n o x y l a n a n d c o u l d not be isolated f r o m each other b y conventional m e t h o d s . T h e s t r u c t u r e s h o w n was d e r i v e d f r o m the analysis of the fragments o b t a i n e d by the e n z y m a t i c d e g r a d a t i o n of t h i s c o m p l e x w i t h the cellulase of T. viride ( x y l o g l u c a n ) , a n d f r o m the a n a l y s i s of the insoluble polysaccharides o b t a i n e d f r o m the controlled S m i t h d e g r a d a t i o n of t h i s f r a c t i o n ( / ? - l , 3 - , l , 4 - g l u c a n ) . F i g u r e 3 shows the s t r u c t u r e of the h e m i c e l l u l o s i c polysaccharides o b t a i n e d f r o m the b r a n cell w a l l p r e p a r a t i o n s . These s t r u c t u r e s were d e d u c e d f r o m the results of the m e t h y l a t i o n a n a l y s i s o f purified fractions (13). In contrast to the e n d o s p e r m cell w a l l , no / ? - l , 3 - , l , 4 - g l u c a n was o b t a i n e d f r o m



P

-(1

6

•

xyi

1 * 2

xyi 6

p

p

Araf

1.4-glucan c)

1

t1

6

xyip

p

p

4)-Xyl -(l h * 4 ) - X y l - ( W 4 ) - X y l - ( l -

6 6

I

xyi p

Jn

4

[1 — f 4 ) -

F i g u r e 2. S t r u c t u r e o f h e m i c e l l u l o s i c polysaccharides o b t a i n e d f r o m the rice e n d o s p e r m cell w a l l , (a) F r o m ref. 12; (b) a n d (c), d e d u c e d f r o m the results described i n ref. 11.

-|>3)-

p

b

xyloglucan >

1 (4-Me-)GlcUA

1

-*4)-Xyl 2

(3) 4 - 1 . 3 - ,

(2)

p

p

1 Ara^

t

4)-Xyl -(1 3

( 1 ) a r a b i n o x y l a n and a r a b i n o g l u c u r o n o x y l a n * )


24.

SHIBUYA

Cell Wall Polymers from Different Parts ofRice

339

(1 ) arabinoga1actoglucuronoxylan ) a

··

>

4)-Xyl -(12 3 t t 1 1 R'R' « 10 ^3

*)-Xyi -0

p

2 t 1 R

3 t 1 R'

P

2 ».


D

P

P

R :

GlcUA -(1-*

R':

periferal residue

or 4-0-Me-GlcUA -(1-

p

p

inner chain sugar residue

sugar

Araf-(1-*

- 4

-*2)-Ara -(lf

14

Gal -(1-* 1.5

-*3)-Ara -(1-* 0.5

Xylp-O-*

->5)-Ara -(1-

n

f

P

f

*2)-Xyl -(1D

(2) xyloglucan ^ 0

Gal. 1 I 2 xyi 1 ' v

t

Xyl 1

*

6 4)-Glc -(l H 4 ) - G l c J2 p

F i g u r e 3. bran,

v

Gal. 1 μ

6 -(1 4)-gv(J^)J5

g

,

v

(

1

]T7

S t r u c t u r e o f h e m i c e l l u l o s i c p o l y s a c c h a r i d e s o b t a i n e d f r o m rice

(a) D e d u c e d f r o m t h e results o f m e t h y l a t i o n a n a l y s i s d e s c r i b e d i n

ref. 13.



340

PLANT C E L L W A L L POLYMERS

the b r a n cell w a l l . T h i s result agreed well w i t h the results of the m e t h y l a t i o n analysis of whole cell w a l l preparations i n w h i c h we c o u l d not detect a n y significant amounts of 1,3-linked glucose residues. T h i s is one of the characteristic features of b r a n hemicelluloses. A g a i n , the acidic a r a b i n o x y l a n was the m a i n c o m p o n e n t of t h i s p r e p a r a t i o n representing over 8 0 % b y weight. However, m e t h y l a t i o n analysis of this a r a b i n o x y l a n i n d i c a t e d a difference i n its detailed s t r u c t u r e c o m p a r e d to the endospermic a r a b i n o x y l a n . For e x a m p l e , the b r a n a r a b i n o x y l a n contained a significant a m o u n t of d o u b l y - b r a n c h e d xylose residues, b u t the e n d o s p e r m a r a b i n o x y l a n d i d not. A l s o , the b r a n a r a b i n o x y l a n seemed to c o n t a i n s o m e w h a t longer, a n d m o r e c o m p l i c a t e d , side-chains t h a n the e n d o s p e r m a r a b i n o x y l a n . T h i s was based u p o n m e t h y l a t i o n a n a l y s i s w h i c h showed the presence of appreciable a m o u n t s of inner c h a i n a r a b i n o s y l residues together w i t h t e r m i n a l g a l a c t o s y l a n d x y l o s y l residues of the b r a n p r e p a r a t i o n . S i m i l a r observations were m a d e for the a r a b i n o x y l a n s isolated f r o m the beeswing b r a n o f wheat (14). B r a n hemicellulose also c o n t a i n e d x y l o g l u c a n as a m i n o r c o m p o n e n t . It was associated w i t h the a r a b i n o x y l a n a n d c o u l d not be isolated b y c o n v e n t i o n a l m e t h o d s . T h e x y l o g l u c a n - r i c h f r a c t i o n was finally digested w i t h a purified arabinofuranosidase a n d an endoxylanase to remove c o n t a m i n a t i n g a r a b i n o x y l a n , a n d the r e s u l t i n g pure x y l o g l u c a n was subjected to m e t h y l a t i o n analysis. U n f o r t u n a t e l y , the s t r u c t u r e of the x y l o g l u c a n so o b t a i n e d ( F i g ure 3) was n o t d i r e c t l y c o m p a r a b l e w i t h the s t r u c t u r e of the e n d o s p e r m x y l o g l u c a n . T h i s was because the s t r u c t u r e i n F i g u r e 3 was deduced f r o m m e t h y l a t i o n analysis of the whole molecule, whereas the e n d o s p e r m x y l o g l u c a n s t r u c t u r e was based u p o n analysis of the fragments l i b e r a t e d by cellulase digestion. Detailed Structure of Side Chains of Bran Arabinoxylan. In order to est a b l i s h the c h e m i c a l i d e n t i t y of the side-chains i n the b r a n a r a b i n o x y l a n , i t was subjected to m i l d a c i d h y d r o l y s i s to liberate oligosaccharides o r i g i n a l l y a t t a c h e d to the x y l a n m a i n c h a i n t h r o u g h the a c i d - l a b i l e a r a b i n o f u r a n o s y l residue (15). A m i x t u r e of the oligosaccharides so o b t a i n e d was reduced a n d m e t h y l a t e d to give the corresponding m e t h y l a t e d oligosaccharide a l d i t o l s . A n a l i q u o t of t h i s m e t h y l a t e d oligosaccharide a l d i t o l m i x t u r e was d i r e c t l y a n a l y z e d by G C - M S , a n d the remainder was h y d r o l y z e d , reduced a n d acetyl a t e d . T h e p r o d u c t s were a n a l y z e d by G C - M S . C h r o m a t o g r a m s of the m e t h y l a t e d oligosaccharide a l d i t o l s showed the presence of d i - a n d t r i s a c charides, i n a d d i t i o n to monosaccharides w h i c h were m a i n l y derived f r o m the n o n - r e d u c i n g end of a r a b i n o f u r a n o s y l residues ( F i g u r e 4). T h e s t r u c ture of each oligosaccharide was d e t e r m i n e d by c o m b i n i n g the i n f o r m a t i o n o b t a i n e d f r o m their C I a n d E I mass s p e c t r a , a n d the linkage i n f o r m a t i o n was o b t a i n e d f r o m the analysis of their h y d r o l y z e d p r o d u c t s . In t h i s way, the s t r u c t u r e of the m a i n peak i n the trisaccharide region (peak 9 i n F i g ure 4) was deduced as follows ( F i g u r e 5). F r o m the pseudo-parent i o n of t h i s peak ( Q M + — M e O H , m / z = 556), it is clear t h a t the o r i g i n a l trisaccharide consists of one hexose a n d two pentose u n i t s . A l s o , f r o m the fragment w i t h m / z = 219, 379 a n d 192, the order of their arrangement m u s t be hexose, pentose, pentose. Hence, the reducing end pentose m u s t be s u b s t i t u t e d at



24.

SHIBUYA

Cell Wall Polymersfrom Different Parts of Rice

341

Monosaccharide

200

400

600

Scan number F i g u r e 4. T o t a l i o n c h r o m a t o g r a m o f the m e t h y l a t e d m o n o / o l i g o s a c c h a r i d e o b t a i n e d b y the p a r t i a l a c i d h y d r o l y s i s o f the b r a n a r a b i n o g a l a c t o g l u curonoxylan.



342

PLANT C E L L W A L L

POLYMERS

F i g u r e 5. P r o p o s e d s t r u c t u r e of peak 9 i n F i g u r e 4 a n d its f r a g m e n t a t i o n p a t t e r n . D e u t e r i u m at C - l p o s i t i o n was i n t r o d u c e d d u r i n g the r e d u c t i o n step w i t h s o d i u m borodeuteride.



24.

Cell Wall Polymers from Different Paris of Rice

SHIBUYA

343

the 0 - 3 p o s i t i o n , j u d g i n g f r o m the f r a g m e n t a t i o n at the reduced a l d i t o l p o r t i o n ( m / z = 45, 46, 89 a n d 90). T o further i d e n t i f y each sugar, a n d also the mode of linkage, the i n f o r m a t i o n o b t a i n e d f r o m the h y d r o l y z e d p r o d u c t s was used. F i r s t l y , the o n l y hexose w i t h a n o n - r e d u c i n g end i n the m i x t u r e of m e t h y l a t e d oligosaccharide a l d i t o l s was galactose; t h a t is, the first hexose u n i t must be galactose. In a s i m i l a r way, the r e d u c i n g end p e n tose m u s t be arabinose, because the o n l y r e d u c i n g e n d pentose s u b s t i t u t e d at 0 - 3 was arabinose. T h e pentose between these two sugar u n i t s s h o u l d be xylose, because the o n l y inner c h a i n pentose residues detected were 4l i n k e d a n d 2 - l i n k e d xylose. A t t h i s p o i n t i t is difficult to select a n y one o f these two p o s s i b i l i t i e s . However, a n e m p i r i c a l rule r e p o r t e d b y K a r k k a i n e n (16) was used to determine the linkage of t h i s i n n e r - c h a i n xylose residue. In t h i s case, fragments derived f r o m the so-called b c A f r a g m e n t a t i o n ( m / z = 379 a n d 347) was m u c h more extensive t h a n those d e r i v e d f r o m b a A f r a g m e n t a t i o n ( m / z = 352 a n d 320), t h u s suggesting a 1,2- rather t h a n a 1,4-linkage. T h u s , the s t r u c t u r e of this oligosaccharide is proposed as the trisaccharide, G a l l , 2 X y l j , l , 3 A r a / . B a s e d u p o n these studies, T a b l e H I s u m m a r i z e s the p a r t i a l l y deduced s t r u c t u r e of the m o n o - , d i - , a n d trisaccharides, o b t a i n e d b y the p a r t i a l d e g r a d a t i o n of the o r i g i n a l a r a b i n o x y l a n . These s t r u c t u r e s m a y not be c o m p l e t e l y a c c u r a t e , because of some u n c e r t a i n t y i n the theory used t o speculate the m o d e of linkage. C o n s e q u e n t l y , each one needs t o be i s o l a t e d to u n a m b i g u o u s l y verify its s t r u c t u r e . These results do, however, p r o v i d e some i n d i c a t i o n of the c o m p l i c a t e d s t r u c t u r e of the side-chains of b r a n arabinoxylan. p

T a b l e I I I . Possible S t r u c t u r e of Oligosaccharides O b t a i n e d by the P a r t i a l A c i d H y d r o l y s i s of R i c e B r a n A r a b i n o g a l a c t o g l u c u r o n o x y l a n Oligosaccharides* 1 2 3 4 5 6 7 8 9 10

Proposed Structure** Pentose ( M a i n l y A r a ) Hexose ( G a l ) Pen-Pen ( X y l - 1 , 3-Ara) Pen-Pen (Xyl-1, 4-Ara) Pen-Pen ( X y l - 1 , 2-Ara) Hex-Pen (Gal-1, 2-Ara) Hex-Pen Hex-Pen Hex-Pen-Pen (Gal-1, 2 - X y l - l , 2-Ara) Pen-Hex-Pen ( X y l - 1 , 3 - G a l - l , 5-Ara)

* See F i g u r e 4. ** P e n = pentose; H e x = hexose.


344



Concluding

Remarks

T h e changes associated w i t h t h e t h i c k e n i n g o f the cell walls i n rice g r a i n are s u m m a r i z e d as follows: (1) a decrease o r s h u t d o w n o f the synthesis o f pectic substances a n d / ? - l , 3 - , l , 4 - g l u c a n ; (2) i n i t i a t i o n o f l i g n i n f o r m a t i o n ; a n d (3) synthesis or a c t i v a t i o n o f the g l y c o s y l transferases responsible for t h e e l o n g a t i o n o f the side chains o f t h e a r a b i n o x y l a n . T h u s , t h e results described i n t h i s article give a n e x a m p l e o f how cell w a l l p o l y m e r s change w i t h differ e n t i a t i o n o f p l a n t tissues, a l t h o u g h a n u n d e r s t a n d i n g o f t h e p h y s i o l o g i c a l m e a n i n g o f these changes requires further studies. Literature Cited

1. Van, K. T. T.; Toubart, P.; Cousson, Α.; Darvil, A. G.; Gollin, D. J.; Chelf, P.; Albersheim, P. Nature 1985, 314, 615-17. 2. Nojiri, H.; Takaku, F.; Terui, Y.; Miura, Y.; Saito, M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 782-86. 3. Albersheim, P.; Darvill, A. G. Sci. Amer. 1985, 253, 44-50. 4. Mares, D. J.; Stone, B.A. Aust. J. Biol. Sci. 1973, 26, 793-812. 5. Selvendran, R. R. Dietary Fibre; Birch, G. G.; Parker, K. J., Eds.; Applied Science: London, 1983; Ch. 7. 6. Tanaka, K.; Yoshida, T.; Asada, K.; Kasai, Z. Arch. Biochem. Bio phys. 1973, 155, 136-43. 7. Shibuya, N.; Nakane, R.; Yasui, Α.; Tanaka, K.; Iwasaki, T. Cereal Chem. 1985, 62, 252-58. 8. Shibuya, N.: Iwasaki, T. Agric. Biol. Chem. 1978, 42, 2259-66. 9. McNeil, M.; Darvill, A. G.; Fry, S. C.; Albersheim, P. Ann. Rev. Biochem. 1984, 53, 625-63. 10. Shibuya, N.; Nakane, R. Phytochem. 1984, 23, 1425-29. 11. Shibuya, N.; Misaki, A. Agric. Biol. Chem. 1978, 42, 2267-74. 12. Shibuya, N.; Misaki, Α.; Iwasaki, T. Agric. Biol. Chem. 1983, 47, 2223-30. 13. Shibuya, N.; Iwasaki, T. Phytochem. 1985, 24, 285-89. 14. Brillouet, J. M.; Joseleau, J. P. Carbohydr. Res. 1987, 159, 109-26. 15. Shibuya, N., unpublished. 16. Karkkainen, J. Carbohydr. Res. 1971, 17, 11-18. RECEIVED March 17, 1989


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