Plant Cell Wall Polymers

Chapter 4 Deposition of C e l l W a l l Components in Conifer Tracheids Keiji Takabe , Kazumi Fukazawa , and Hiroshi Harada 1

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1Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, 2

Sapporo, Japan Rector Office, Kasetsart University, Bangkhen, Bangkok, 10900, Thailand

The cell wall deposition processes, distribution of cell wall components, and cell organellae involved in the biosynthesis of polysaccharides and lignin in conifer tracheids were investigated using several chemical and microscopy techniques. The deposition process for cellulose was found to differ from that of hemicelluloses. Cellulose deposited actively between the S and S developmental stages, especially in the middle part of the S stage. On the other hand, mannans and xylans were laid down between the latter part of S and the early part of S development, and between the latter part of the S and S stages. These results suggest that (i) the middle portion of S is rich in cellulose, and (ii) hemicelluloses are abundant in S , and the outer and inner portions of S and S tissue. Lignification was initiated at the outer surface of the primary wall cell corners, and proceeded into the intercellular layers, and the intercellular substances between cell corners. Lignification of the secondary walls was initiated at the S cell corner, then proceeded to the unlignified S layer and toward the lumen, while lagging behind cell wall thickening. Cellulose synthesis occurred at the plasma membrane. The Golgi-body, and a small circular vesicle derived from the rough endoplasmic reticulum, were involved in the biosynthesis and/or transport of hemicelluloses, while the Golgi-body and smooth-endoplasmic reticulum were involved in the biosynthesis and/or transport of monolignols. 1

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0097~6156/89/0399-0047$06.00A) © 1989 American Chemical Societv

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48

PLANT C E L L W A L L POLYMERS

C e l l u l o s e , hemicelluloses, a n d l i g n i n are the m a i n c o m p o n e n t s of cell walls i n w o o d y p l a n t s . For a l o n g t i m e , these p l a n t p o l y m e r s have s t i m u l a t e d the interest o f m a n y p l a n t botanists a n d biochemists i n terms of their b i o s y n thetic pathways, functional interrelationships, and anatomical distribution. T h e cell w a l l of conifer tracheids consists of b o t h p r i m a r y a n d seco n d a r y w a l l s . T h e p r i m a r y w a l l is formed d u r i n g cell d i v i s i o n a n d s u b sequent cell enlargement, whereas secondary w a l l f o r m a t i o n o n l y occurs after cell enlargement has been c o m p l e t e d . Secondary walls are s u b d i v i d e d i n t o three layers, n a m e d S i , S2, a n d S3, respectively. A large effort has been devoted to e l u c i d a t i n g (i) s t r u c t u r a l a n d c h e m i c a l properties o f the polysaccharides a n d l i g n i n i n each cell w a l l layer a n d their f u n c t i o n a l i n t e r r e l a t i o n s h i p s , a n d (ii) the role of cell organellae i n cell w a l l biosynthesis. I n spite of t h i s , our knowledge of the entire process of cell w a l l c o n s t r u c t i o n a n d the i n t e r r e l a t i o n s h i p s of cell w a l l c o m p o n e n t s s t i l l r e m a i n s i n c o m p l e t e . Deposition of Polysaccharides P i o n e e r i n g w o r k o n p o l y s a c c h a r i d e d i s t r i b u t i o n i n the cell w a l l was carried out b y M e i e r a n d W i l k i e (1) a n d M e i e r (2). T h e y isolated r a d i a l sections f r o m the differentiating x y l e m of Pinus sylvestris, Picea abies, a n d Betula verrucosa, separated each i n t o s u b c e l l u l a r fractions b y a m i c r o m a n i p u l a t o r , a n d a n a l y z e d the monosaccharide c o m p o s i t i o n of the different p o l y s a c c h a r i d e fractions b y paper c h r o m a t o g r a p h y . F r o m these studies i t was concluded t h a t , i n softwood tracheids, the outer p a r t of the S2 layer was richest i n cellulose, whereas the S3 layer was richest i n g l u c u r o n o a r a b i n o x y l a n a n d the g l u c o m a n n a n content g r a d u a l l y increased towards the l u m e n . O n the other h a n d , i n h a r d w o o d fibers, the inner p a r t of S a n d S3 were richest i n cellulose, w h i l e S i a n d the outer p a r t of S2 showed a h i g h g l u c u r o n o x y l a n content. Côté et al. (3) also investigated the p o l y s a c c h a ride d i s t r i b u t i o n i n Abies balsamea tracheids a c c o r d i n g to the m e t h o d of M e i e r a n d o b t a i n e d s i m i l a r conclusions. 2

L a r s o n (4,5) fed C 0 2 p h o t o s y n t h e t i c a l l y to Pinus resinosa, d i v i d e d the differentiating x y l e m i n t o several fractions, a n d c o u n t e d the r a d i o a c t i v i t y of each cell w a l l c o m p o n e n t . F r o m these studies, i t was c o n c l u d e d t h a t as t r a c h e i d m a t u r a t i o n o c c u r r e d , xylose d e p o s i t i o n increased, whereas mannose r e m a i n e d r e l a t i v e l y c o n s t a n t , a n d b o t h arabinose a n d galactose decreased considerably. 1 4

In recent years, H a r d e l l a n d W e s t e r m a r k (6) scratched Picea abies t r a cheids w i t h tweezers, collected i n d i v i d u a l cell w a l l layers, a n d then a n a l y z e d the average monosaccharide c o m p o s i t i o n . S u r p r i s i n g l y , a m o n g the i n d i v i d u a l cell w a l l layers no significant difference i n the mannose:xylose:glucose r a t i o a m o n g i n d i v i d u a l cell w a l l layers was observed. W e have also s t u d i e d polysaccharide d e p o s i t i o n processes d u r i n g cell w a l l f o r m a t i o n (7), b y g a s - l i q u i d c h r o m a t o g r a p h i c a n a l y s i s of fractions sep-

4.

T A K A B E et A L .

Cell Wall Components in Conifer Tracheids

a r a t e d b y m i c r o f r a c t i o n a t i o n a n d enriched i n cells at different t a l stages. ria japonica

49 developmen-

I n t h a t i n v e s t i g a t i o n , the d i f f e r e n t i a t i n g s y s t e m of

Cryptome-

was separated i n t o twelve fractions a n d each h a d its n e u t r a l

monosaccharide

content a n d t y p e d e t e r m i n e d ( F i g s . 1 a n d 2). ( N o t e t h a t

glucose, m a n n o s e , a n d xylose are m a i n l y d e r i v e d f r o m cellulose, m a n n a n s , a n d x y l a n s , respectively.) T h u s , i t was c o n c l u d e d t h a t cellulose was m a i n l y deposited i n the m i d d l e p a r t of the S2 layer, whereas hemicelluloses

(man-

n a n s , x y l a n ) were f o u n d m a i n l y i n the S i a n d the outer p a r t of the S2 a n d S3 layers ( F i g . 3). T h e s e results were f u r t h e r c o n f i r m e d f r o m o u r t r a n s m i s s i o n electron m i c r o s c o p y

( T E M ) studies o n d i f f e r e n t i a t i n g x y l e m s t a i n e d

w i t h P A T A g . T h i s technique, developed by Thiéry (8), is specific for h e m i celluloses a n d showed h e a v y s t a i n i n g o f the S i , outer S2 a n d S3 layers. Interestingly, the w a r t y layer was also h e a v i l y s t a i n e d b y P A T A g , i n d i c a t i n g i t to be m a i n l y c o m p o s e d of hemicelluloses. W e next investigated the i n c o r p o r a t i o n of U w a l l p o l y m e r s d u r i n g cell w a l l f o r m a t i o n (9).

1 4

C glucose i n t o the cell

A f t e r i n c o r p o r a t i o n o f the

l a b e l l e d sugar, the differentiating x y l e m of Cryptomeria

japonica

was sepa-

r a t e d i n t o 8 f r a c t i o n s , each of w h i c h was subjected to m i l d a c i d h y d r o l y s i s . T h e monosaccharides, so released f r o m the polysaccharides, were t h e n sepa r a t e d b y t h i n - l a y e r c h r o m a t o g r a p h y (tic) a n d the i n d i v i d u a l r a d i o a c t i v i t y content for each sugar was v i s u a l i z e d b y a u t o r a d i o g r a p h y a n d by scintillation counting.

measured

A s can be seen f r o m F i g u r e 4, the d i s t r i b u t i o n

o f r a d i o a c t i v i t y i n t o the i n d i v i d u a l sugars was i n g o o d agreement w i t h o u r previous analyses of the p o l y m e r s , i.e., cellulose d e p o s i t i o n m a i n l y o c c u r r e d i n the m i d d l e p a r t of the S2 to S3 d e v e l o p m e n t a l stage, whereas x y l a n dep o s i t i o n was i n the S i t o e a r l y S2 a n d a g a i n i n the S3 d e v e l o p m e n t a l layers. M a n n a n d e p o s i t i o n o c c u r r e d m a i n l y d u r i n g secondary w a l l f o r m a t i o n r a t h e r t h a n d u r i n g f o r m a t i o n of the p r i m a r y w a l l . Lignification of Tracheids T h e first s t u d y o n l i g n i f i c a t i o n of conifer W a r d r o p (10). radiata

tracheids was c a r r i e d out

F r o m e x a m i n a t i o n of the d i f f e r e n t i a t i n g x y l e m of

by

Pinus

under a n u l t r a v i o l e t microscope, he observed t h a t l i g n i f i c a t i o n was

i n i t i a t e d at the cell corners of the p r i m a r y w a l l , then extended t o the m i d dle l a m e l l a a n d secondary

wall.

I m a g a w a et al.

(11) subsequently

U V - p h o t o m i c r o g r a p h s of the differentiating x y l e m of Larix d e m o n s t r a t e d t h a t l i g n i f i c a t i o n proceeded as follows:

leptolepis

took and

lignin accumulation

begins i n the i n t e r c e l l u l a r layer at the cell corners a n d p i t b o r d e r s ,

then

extends to b o t h r a d i a l a n d t a n g e n t i a l m i d d l e l a m e l l a , a n d t h e n towards the l u m e n . F u j i t a et al. (12) investigated l i g n i f i c a t i o n of Cryptomeria

japonica

compression w o o d b y the same m e t h o d . These a u t h o r s f o u n d t h a t there are two types of l i g n i n d e p o s i t i o n processes: O n e was p r i m a r y w a l l l i g n i f i c a t i o n w h i c h o c c u r r e d f r o m the e a r l y phase of S i d e p o s i t i o n to the early phase of


50

F i g u r e 1. C h a n g e s i n the absolute a m o u n t of sugars w i t h t r a c h e i d m a t u r a t i o n . T h e various components are designated as follows: glucose, • ; m a n n o s e , A ; xylose, Δ ; arabinose, Q î d galactose, φ . a

n

4.

TAKABE ET A L


51

F i g u r e 2. T h e a m o u n t of increase i n sugars (the difference o f the a b s o l u t e a m o u n t s of sugar between the n e i g h b o r i n g f r a c t i o n s ) . T h e dashed l i n e shows the s u m of arabinose, galactose, x y l o s e , a n d mannose.

PLANT C E L L W A L L P O L Y M E R S

52

(%) 100Gal.

80H Ara.

i i i i f i Ëlfllil

Xyl. 60-

Mit

Man.

AOH Glc

20·

1 • p-

2 —

2-3 Si

3-4 1

4-5

5-6 S2

6-7

7-8 1

8-9 S3

—

F i g u r e 3. D i s t r i b u t i o n of polysaccharides t h r o u g h the cell w a l l . C , cellulose; M , galacto-glucomannan; X , arabino-4-O-methylglucuronoxylan.

4.

TAKABE ET A L

u


1

2

F i g u r e 4. T h e c o m p o s i t i o n

3 4 5 6 Fraction Number

7

53

8

o f r a d i o a c t i v i t y i n n e u t r a l sugars. A r a b i c n u -

merals are the f r a c t i o n n u m b e r s . T h e differentiating stages i n each f r a c t i o n are as follows: F r a c t i o n s 1-2, p r i m a r y w a l l stage; 3, S i stage; 4-6, S2 stage; 7-8, S 3 stage.

54


S2 t h i c k e n i n g ; the other was secondary w a l l l i g n i f i c a t i o n w h i c h proceeded after S2 t h i c k e n i n g . A l t h o u g h U V - m i c r o s c o p y has p r o v i d e d m u c h i n f o r m a t i o n o n the l i g n i fication process a n d l i g n i n d i s t r i b u t i o n t h r o u g h the cell w a l l , i t has been o f l i m i t e d value because o f i t s low r e s o l v i n g power as c o m p a r e d t o electron microscopy. C o n s e q u e n t l y , some workers, u s i n g specimens fixed w i t h p o t a s s i u m p e r m a n g a n a t e , s t u d i e d l i g n i f i c a t i o n u s i n g T E M . I n t h i s way, W a r d r o p ( 1 3 , 1 4 ) f o u n d t h a t w i t h Eucalyptus elaeophora, l i g n i n f o r m a t i o n was i n i t i ated at the m i d d l e l a m e l l a o f the cell corners, a n d subsequently at the outer p a r t of the S i layer. It t h e n proceeded a l o n g the m i d d l e l a m e l l a , t h r o u g h the p r i m a r y w a l l , a n d u l t i m a t e l y to the secondary w a l l . K u t s c h a a n d S c h w a r z m a n n (15) also e x a m i n e d the l i g n i f i c a t i o n of Abies balsamea tracheids b y T E M a n d showed t h a t it was i n i t i a t e d i n the m i d d l e l a m e l l a between p i t borders of adjacent t r a c h e i d s , a n d t h e n e x t e n d e d i n t o the p i t borders a n d cell corners. I n cell corners, i t o c c u r r e d at either the outer p o r t i o n o f the p r i m a r y w a l l or the m i d d l e l a m e l l a . A f t e r t h a t , l i g n i f i c a t i o n t o o k place i n the cell corner region of the S i layer, l e a v i n g the p r i m a r y w a l l u n l i g n i f i e d , a n d t h e n proceeded subsequently t o w a r d the l u m e n . T h i s technique, u s i n g p e r m a n g a n a t e fixation, c a n , however, cause s w e l l i n g of b o t h the cell a n d the cell w a l l , a n d the e x t r a c t i o n of m a n y cell c o m p o n e n t s . Indeed, K i s h i et ai (16) r e p o r t e d t h a t the s t a i n i n g i n t e n s i t y p r o d u c e d b y p e r m a n g a n a t e does not reflect true l i g n i n content, t h u s l e a v i n g the aforesaid results i n some d o u b t . S a k a a n d T h o m a s (17) also investigated the l i g n i f i c a t i o n of Pinus taeda tracheids b y the S E M - E D X A technique, a n d showed t h a t i t was i n i t i a t e d i n the cell corner m i d d l e l a m e l l a a n d c o m p o u n d m i d d l e l a m e l l a regions d u r i n g S i f o r m a t i o n . Subsequently, r a p i d l i g n i n d e p o s i t i o n o c c u r r e d i n b o t h regions. Secondary w a l l l i g n i f i c a t i o n was i n i t i a t e d w h e n the m i d d l e l a m e l l a l i g n i n c o n c e n t r a t i o n approached 5 0 % of its m a x i m u m , a n d t h e n proceeded towards the l u m e n . W e (18-20) have also investigated the l i g n i f i c a t i o n process u s i n g Cryptomeria japonica tracheids. Techniques employed were a d m i n i s t r a t i o n o f t r i t i a t e d p h e n y l a l a n i n e as a l i g n i n precursor, followed b y a c o m b i n a t i o n of U V - m i c r o s c o p y , light microscopic a u t o r a d i o g r a p h y , a n d T E M coupled w i t h a p p r o p r i a t e c h e m i c a l t r e a t m e n t s of u l t r a - t h i n sections. F i g u r e 5 shows densitometer traces o f U V - p h o t o n e g a t i v e s of differentiating x y l e m . T h e U V - a b s o r p t i o n of the c o m p o u n d m i d d l e l a m e l l a was first detected i n the t r a c h e i d at the S i d e v e l o p m e n t a l stage, t h e n increased d u r i n g secondary w a l l t h i c k e n i n g , b e c o m i n g constant after the S 3 stage. O n the other h a n d , U V - a b s o r p t i o n at the secondary w a l l was first observed at the outer p o r t i o n i n the t r a c h e i d of the S2 stage, t h e n spread slowly towards the l u m e n i n subsequent stages. T h e t r a c h e i d i n the final p a r t of cell w a l l f o r m a t i o n showed u n i f o r m a b s o r p t i o n t h r o u g h the secondary w a l l . F r o m F i g u r e 6,

4.


55


F i g u r e 5. D e n s i t o m e t e r traces of U V - p h o t o n e g a t i v e s . A r a b i c n u m e r a l s i n dicate the cell n u m b e r w h i c h s t a r t s f r o m the cell j u s t before S i f o r m a t i o n .

. 0

5

10

15

20

.?r - -. 25

30

Cell

ι • 0

. 5

Ο

• 15

20

,·,.... 25

30

Number

F i g u r e 6. I n c o r p o r a t i o n of t r i t i a t e d p h e n y l a l a n i n e i n t o the c o m p o u n d

mid

dle l a m e l l a l i g n i n a n d the secondary w a l l l i g n i n d e t e r m i n e d b y c o u n t i n g the silver g r a i n s . S y m b o l s are as follows: φ , c o m p o u n d m i d d l e l a m e l l a ; 0 > secondary w a l l .

56


it is evident t h a t the labelled l i g n i n precursor was r a p i d l y i n c o r p o r a t e d i n t o the c o m p o u n d m i d d l e l a m e l l a l i g n i n , whereas i t entered s l o w l y i n t o secondary w a l l l i g n i n . A d d i t i o n a l l y , i n c o r p o r a t i o n i n t o c o m p o u n d m i d d l e l a m e l l a l i g n i n took place d u r i n g S i a n d S2 d e v e l o p m e n t a l stages, w h i l e i n c o r p o r a t i o n i n t o secondary w a l l l i g n i n o c c u r r e d m a i n l y after the S3 stage. In the l a t t e r case, r a d i o a c t i v i t y was d i s t r i b u t e d t h r o u g h o u t the secondary w a l l . T h i s i n d i c a t e d t h a t m o n o l i g n o l s were c o n t i n u o u s l y s u p p l i e d to a l l a r eas o f the secondary w a l l . T h u s , the l i g n i n content i n the secondary w a l l g r a d u a l l y increased b y repeated l i n k i n g of m o n o l i g n o l r a d i c a l s . These findings were also s u p p o r t e d b y analysis of the l i g n i n skeleton of the d i f f e r e n t i a t i n g x y l e m , o b t a i n e d b y t r e a t m e n t of u l t r a - t h i n sections w i t h h y d r o f l u o r i c a c i d after resin e x t r a c t i o n (20). T h i s removes polysaccharides effectively w i t h o u t a n y s w e l l i n g of the cell w a l l . In t h i s way, we f o u n d t h a t l i g n i f i c a t i o n i n the Cryptomeria japonica t r a c h e i d was i n i t i a t e d at the outer surface o f the p r i m a r y w a l l i n the cell corners, j u s t before S i f o r m a t i o n . Subsequently, i t proceeded to the i n t e r c e l l u l a r layer together w i t h l i g n i n d e p o s i t i o n i n the i n t e r c e l l u l a r substances between the cell corners. W h e n the t r a c h e i d was adjacent to a r a y p a r e n c h y m a , the cell corner region o n the r a y p a r e n c h y m a side was lignified earlier t h a n t h a t o n the opposite one. It was very i n t e r e s t i n g t h a t secondary w a l l l i g n i f i c a t i o n was also i n i t i a t e d at the S i cell corner region d u r i n g the S i d e v e l o p m e n t a l stage. It t h e n proceeded to the u n l i g n i f i e d S i layer. W h e n the t r a c h e i d was adjacent to a r a y p a r e n c h y m a , l i g n i f i c a t i o n of the S i layer was also earlier o n the r a y side. A f t e r t h a t , l i g n i f i c a t i o n g r a d u a l l y spread towards the l u m e n , l a g g i n g b e h i n d cell w a l l t h i c k e n i n g . L i g n i n d e p o s i t i o n t h e n p r e d o m i n a t e d after the S3 stage, w h i l e less active d u r i n g the S i , S2, a n d S3 d e v e l o p m e n t a l stages. T h e l i g n i n content of the secondary w a l l became f a i r l y constant i n the final stage of cell w a l l f o r m a t i o n , t h o u g h the w a r t y layer was more h i g h l y lignified. Changes in C e l l Organellae D u r i n g C e l l W a l l F o r m a t i o n T h e cell organellae i n w o o d y plants are the nucleus, m i t o c h o n d r i o n , r o u g h - e n d o p l a s m i c r e t i c u l u m ( r - E R ) , s m o o t h e n d o p l a s m i c r e t i c u l u m (sE R ) , G o l g i - b o d y , p l a s t i d , vacuole, m i c r o b o d y , etc. T h e i r functions are very c o m p l i c a t e d , a n d some have definite roles i n the biosynthesis o f c e l l - w a l l components. H e n c e , changes i n size of cell organellae are likely to o c c u r , since c e l l - w a l l c o m p o s i t i o n depends u p o n the stage of w a l l development. W e t r i e d to estimate the size of the cell organellae i n the c y t o p l a s m , e x c l u d i n g the vacuolar c o m p a r t m e n t . T o do this, we took at r a n d o m a few h u n d r e d electron m i c r o g r a p h s of cells i n the differentiating x y l e m a n d measured the area o f each cell organelle i n the c y t o p l a s m b y a d i g i t i z e r c o u p l e d to a m i c r o c o m p u t e r . W e f o u n d not o n l y changes i n the area of cell organellae i n the c y t o p l a s m , b u t also i n their s t r u c t u r e d u r i n g cell

4.

TAKABE ET A L

wall formation.

57


F i g u r e 7 shows the results o f s e m i - q u a n t i t a t i v e a n a l y s i s

of the areas for each cell o r g a n e l l a s d u r i n g cell w a l l f o r m a t i o n .

It was

s u r p r i s i n g t h a t the G o l g i - b o d y , r - E R , a n d s - E R , showed a p p r e c i a b l e changes i n area d u r i n g cell w a l l f o r m a t i o n . T h e area o f the G o l g i - b o d y was largest at the S i stage, a n d t h e n g r a d u a l l y decreased i n size w i t h m a t u r a t i o n o f the t r a c h e i d , whereas the r - E R was largest i n the p r i m a r y w a l l stage, a n d t h e n g r a d u a l l y decreased w i t h cell w a l l f o r m a t i o n . T h e s - E R , o n the other h a n d , was a m i n o r organelle f r o m the p r i m a r y w a l l stage t o the e a r l y p a r t o f the S2 stage, a n d t h e n showed a g r a d u a l increase i n a r e a t o w a r d the S3 d e v e l o p m e n t a l stage. T h e m o s t s t r i k i n g fact was t h a t the e n l a r g e m e n t o f the s - E R c o i n c i d e d w i t h t h a t o f active l i g n i f i c a t i o n of the secondary w a l l . F i g u r e 8 shows the changes i n the s t r u c t u r e o f cell o r g a n e l l a s .

The

p h o t o g r a p h s are t y p i c a l s t r u c t u r e s i n each d i f f e r e n t i a t i n g stage. N o t e t h a t the G o l g i - b o d y consists of t h i n c e n t r a l cisternae a n d r e l a t i v e l y s m a l l vesicles d u r i n g the p r i m a r y w a l l stage.

T h e c e n t r a l cisternae t h e n b e c o m e t h i c k ,

t h i s b e i n g a c c o m p a n i e d b y the f o r m a t i o n o f m a n y large vesicles c o n t a i n i n g fibrillar

m a t e r i a l d u r i n g the S i a n d S2 d e v e l o p m e n t a l stages.

After that,

the c e n t r a l cisternae b e c a m e s m a l l i n size, t h o u g h the t h i c k n e s s was s i m i l a r t o t h a t o f p r e v i o u s stages. Interestingly, t h i s stage appears to be a c c o m p a n i e d b y the f o r m a t i o n of o n l y a few vesicles, i n d i c a t i n g depression of G o l g i activity. S e v e r a l r e t i c u l a of the r - E R show a n ordered a r r a n g e m e n t a n d m a n y ribosomes are a t t a c h e d to t h e i r m e m b r a n e d u r i n g the p r i m a r y w a l l d e v e l o p m e n t stage. A s m a t u r a t i o n proceeds, the r - E R ' s t h e n g r a d u a l l y decrease not o n l y i n n u m b e r a n d l e n g t h of r e t i c u l a , b u t also i n the n u m b e r o f r i b o somes. T h e s - E R ' s , o n the other h a n d , b e c o m e largest after the S3 stage, a n d sometimes a t t a c h ribosomes at their t e r m i n a l s . D u r i n g p r i m a r y w a l l f o r m a t i o n the p l a s t i d s c o n t a i n s t a r c h a n d other m a t e r i a l s w h i c h s t a i n h e a v i l y w i t h u r a n y l acetate a n d l e a d c i t r a t e . W h e n the t r a c h e i d s t a r t s to f o r m the S i layer, the p l a s t i d becomes s u r r o u n d e d b y a n e n d o p l a s m i c r e t i c u l u m . W h i l e the fate of these c o m p o u n d s is u n k n o w n , it c a n be envisaged t h a t they are used for generation o f energy a n d / o r a source o f cell w a l l m a t e r i a l s . C e l l Organellae Involved in Biosynthesis of Polysaccharides T h o u g h cellulose is one of the most i m p o r t a n t b i o p o l y m e r s , i t has not yet been possible to c o m p l e t e l y e l u c i d a t e i t s b i o s y n t h e t i c p a t h w a y , or e s t a b l i s h e x a c t l y the cell organellae i n v o l v e d i n its synthesis. H o w e v e r , d u r i n g the last decade, the freeze fracture technique has been a p p l i e d to investigate cell w a l l f o r m a t i o n , a n d t h i s has p r o d u c e d m u c h i n f o r m a t i o n o n the site where cellulose synthesis occurs.

It is now generally accepted t h a t b o t h t e r m i -

n a l a n d rosette complexes are responsible for cellulose synthesis (21). O u r results (19,22) s u p p o r t t h a t v i e w .

In a T E M - a u t o r a d i o g r a p h i c investiga-

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F i g u r e 7. S e m i - q u a n t i t a t i v e measurements of cell organellae i n the c y t o plasm.

4.



59

F i g u r e 8. C h a n g e s i n the s t r u c t u r e of cell organellae d u r i n g cell w a l l form a t i o n . U p p e r , m i d d l e , a n d lower p h o t o g r a p h s are G o l g i - b o d y , r - E R , a n d p l a s t i d , respectively. A b b r e v i a t i o n s are as follows: P , p r i m a r y w a l l stage; S2#, e a r l y p a r t of S2 stage; S 2 L , later p a r t of S2 stage.

60


t i o n of differentiating x y l e m , p r e v i o u s l y a d m i n i s t e r e d t r i t i a t e d glucose, the r a d i o a c t i v i t y was concentrated at the b o u n d a r y between the n e w l y f o r m e d c e l l - w a l l a n d the c y t o p l a s m i n the m i d d l e a n d l a t t e r p a r t s of the S2 stage. A c o m p a r i s o n w i t h F i g u r e 4 indicates t h a t a m a j o r c o m p o n e n t of the l a b e l l e d m a t e r i a l s i n these areas was i n the cellulose p o l y m e r , a n d hence glucose derived. F r o m a cytochemical investigation, fibrillar materials having a w i d t h of 6-7 m m were sometimes observed, these b e i n g generated f r o m the p l a s m a m e m b r a n e ( F i g . 9). T h e y h a d s i m i l a r dimensions to cellulose m i c r o f i b r i l s f r o m gelatinous layers of Populus euramericana as s h o w n b y S u g i y a m a et ai (23), thereby i n d i c a t i n g the involvement of the p l a s m a m e m b r a n e i n cellulose synthesis. T h e biosynthesis of cell w a l l polysaccharides has also been s t u d i e d b y c y t o c h e m i c a l s t a i n i n g m e t h o d s . P A T A g s t a i n i n g (8) resulted i n e s t a b l i s h i n g cell organellae i n v o l v e d i n the biosynthesis of m a n y polysaccharides. P i c k e t t - H e a p s (24) a d a p t e d t h i s s t a i n i n g procedure to the root t i p s a n d coleoptiles of Triticum vulgare seedlings. I n t h i s way, he showed t h a t G o l g i cisternae a n d their vesicles were s t a i n e d positively, whereas E R cisternae s t a i n e d negatively. T h i s led to the conclusion t h a t G o l g i - b o d i e s were i n v o l v e d i n the biosynthesis a n d / o r t r a n s p o r t of polysaccharides. T h e s e results were confirmed a g a i n i n later studies b y Fowke a n d P i c k e t t - H e a p s (25) a n d R y s e r (26). I n recent years, S u g i y a m a et ai (27) observed the Valonia macrophysa c e l l - w a l l b y means of selective v i s u a l i z a t i o n a n d c h e m i c a l a n a l ysis of c e l l - w a l l components. T h e authors f o u n d t h a t the m a t e r i a l s s t a i n e d p o s i t i v e l y w i t h P A T A g , or a c o m b i n a t i o n of u r a n y l acetate a n d lead c i t r a t e , were non-cellulosic polysaccharides. W e have also a p p l i e d the P A T A g s t a i n i n g to the d i f f e r e n t i a t i n g x y l e m of Cryptomeria japonica (22). W h i l e the contents i n the Golgi-vesicles s t a i n e d p o s i t i v e l y at a l l stages of cell w a l l f o r m a t i o n , there were three m a i n observ a t i o n s . D u r i n g p r i m a r y w a l l f o r m a t i o n , the Golgi-vesicles were s m a l l a n d their contents o n l y s t a i n e d weakly. A f t e r t h a t , the vesicles b e c a m e larger a n d the c o m p o n e n t s w h i c h showed f i b r i l l a r s t r u c t u r e s were s t r o n g l y s t a i n e d . F o l l o w i n g the S 3 stage, the contents a g a i n s t a i n e d weakly, a n d h a d a s l i m y appearance. T h i s p r e s u m a b l y (i) reflects changes i n non-cellulosic p o l y s a c charide c o m p o s i t i o n a n d (ii) suggests t h a t the G o l g i - b o d i e s are i n v o l v e d i n the biosynthesis of non-cellulosic polysaccharides. F i g u r e 10 shows s m a l l c i r c u l a r vesicles w h i c h were d i s t r i b u t e d at the e n d o f the r - E R cisternae a n d between the cisternae, a n d w h i c h were sometimes a t t a c h e d to the E R m e m b r a n e . A s the size a n d the shape of these vesicles (75 n m i n m e a n diameter) were different f r o m those of Golgi-vesicles (130 n m i n m e a n d i a m e t e r ) , the s m a l l c i r c u l a r vesicles were p r e s u m a b l y der i v e d f r o m r - E R ; they also s t a i n e d p o s i t i v e l y w i t h P A T A g . These facts suggest t h a t the s m a l l c i r c u l a r vesicles f r o m the E R are i n v o l v e d i n the biosynthesis a n d / o r t r a n s p o r t of non-cellulosic polysaccharides.

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TAKABE ET A L


61

F i g u r e 9. A t r a c h e i d i n the S3 stage. F i b r i l l a r m a t e r i a l s (arrows) are generated f r o m the p l a s m a m e m b r a n e . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 22. ©

1986, J a p a n W o o d Research Society.)

62


F i g u r e 10. A differentiating t r a c h e i d stained w i t h P A T A g . S m a l l c i r c u l a r vesicles (arrowheads), d i s t r i b u t e d near the E R are s t a i n e d positively. T h e Golgi-vesicles are also s t a i n e d positively. A b b r e v i a t i o n s are as follows: G V , G o l g i - v e s i c l e ; E R , e n d o p l a s m i c r e t i c u l u m . Scale bar is 5 0 0 n m . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 22. ©

1986, J a p a n W o o d Research Society.)

4.

TAKABE ET AL.


63

C e l l Organellae Involved in the Biosynthesis of L i g n i n O n l y a few workers have a t t e m p t e d to i d e n t i f y the cell organellae i n v o l v e d i n the biosynthesis of l i g n i n . P i c k e t t - H e a p s (28) observed l i g n i f i c a t i o n i n the x y l e m w a l l of wheat coleoptiles a n d suggested the i n v o l v e m e n t of b o t h the G o l g i - b o d i e s a n d r - E R , since r a d i o a c t i v i t y was d i s t r i b u t e d o n b o t h o r g a n e l lae. M o r e recently, F u j i t a et al. (29) s t u d i e d l i g n i f i c a t i o n i n c o m p r e s s i o n w o o d cell walls of Cryptomeria

japonica

by T E M - a u t o r a d i o g r a p h y . Like

P i c k e t t - H e a p s , they c o n c l u d e d t h a t the G o l g i - b o d i e s p a r t i c i p a t e d i n l i g n i n biosynthesis. W a r d r o p ( 1 3 , 1 4 ) e x a m i n e d s c l e r e n c h y m a fibers of Liriodendron if era a n d sclereids of Pyrus

communis,

tulip-

p r e v i o u s l y fixed w i t h KMnÛ4, a n d

c o n c l u d e d t h a t the vesicles s u p p l i e d t h e i r contents to the cell w a l l , t h o u g h the o r i g i n of the vesicles was not e s t a b l i s h e d . A s discussed before, however, p e r m a n g a n a t e fixation is u n d e s i r a b l e for these c y t o l o g i c a l o b s e r v a t i o n s . C o n s e q u e n t l y , we a d m i n i s t e r e d t r i t i a t e d p h e n y l a l a n i n e , a l i g n i n p r e c u r sor, t o the d i f f e r e n t i a t i n g x y l e m o f Cryptomeria

japonica,

and determined

the l o c a t i o n of the l a b e l b y T E M - a u t o r a d i o g r a p h y (30). T h e r a d i o a c t i v i t y was l o c a t e d o n the c o m p o u n d m i d d l e l a m e l l a , i n c l u d i n g the cell-corner regions f r o m the final p a r t of the p r i m a r y w a l l stage to the e a r l y p a r t of the S2 d e v e l o p m e n t a l stage. C o r r e s p o n d i n g to these stages, the r a d i o a c t i v i t y was d i s t r i b u t e d on G o l g i - b o d i e s w h i c h secreted m a n y vesicles, a n d r - E R ' s ( F i g . 11). R a d i o a c t i v i t y was a b u n d a n t l y l o c a t e d o n the secondary w a l l f r o m the S3 stage to the c o m p l e t i o n of secondary w a l l l i g n i f i c a t i o n . R a d i o a c t i v i t y was also l o c a t e d o n s - E R ' s ( F i g . 12). T h e r a d i o a c t i v i t y o n the cell w a l l coi n c i d e d w i t h l i g n i n d e p o s i t i o n , as e v i d e n c e d b y U V - m i c r o s c o p y a n d T E M . T h e s e results therefore suggest t h a t the G o l g i - b o d y , r - E R , a n d s - E R are a l l i n v o l v e d i n the biosynthesis of l i g n i n . However, since i t is w e l l k n o w n t h a t the r - E R is a site of p r o t e i n synthesis, some p h e n y l a l a n i n e m a y be used for this purpose.

O n the other h a n d , the G o l g i - b o d i e s secrete m a n y vesicles

d u r i n g active l i g n i f i c a t i o n of the c o m p o u n d m i d d l e l a m e l l a a n d secondary wall.

T h e a d m i n i s t e r e d l i g n i n precursor may, therefore, be i n c o r p o r a t e d

i n t o the G o l g i - b o d y d i r e c t l y or v i a other organellae, a n d t h e n converted i n t o m o n o l i g n o l s v i a e n z y m a t i c conversion. T h e m o n o l i g n o l s c a n t h e n be secreted i n t o the cell w a l l b y exocytosis of the G o l g i - v e s i c l e s . M a n y s - E R ' s also a p p e a r i n the c y t o p l a s m a n d these sometimes fuse to the p l a s m a m e m b r a n e at the S3 stage. T h u s , l i g n i n precursors m a y also be converted i n t o m o n o l i g n o l s at the l u m e n or the m e m b r a n e of s - E R ' s , a n d t h e n secreted i n t o the cell w a l l b y fusion of s - E R to the p l a s m a m e m b r a n e .

64


F i g u r e 11. A t r a c h e i d i n the early part of S2 stage. R a d i o a c t i v i t i e s observed on the G o l g i - b o d i e s a n d c o m p o u n d m i d d l e l a m e l l a .

are

F i g u r e 12. A t r a c h e i d after S stage. C y t o p l a s m is filled w i t h s - E R ' s . R a d i o a c t i v i t y is d i s t r i b u t e d on the s - E R ' s a n d secondary w a l l . 3

4.

TAKABE ET A L

65


Concluding Remarks It is e v i d e n t t h a t d e p o s i t i o n processes for cellulose a n d hemicelluloses i n conifer tracheids are q u i t e different, i.e., cellulose is m a i n l y d e p o s i t e d i n t h e m i d d l e p a r t o f the S2 d e v e l o p m e n t a l stage, whereas h e m i c e l l u l o s e d e p o s i t i o n o c c u r s f r o m the S i t o the e a r l y p a r t o f t h e S2 stage, a n d t h e n d u r i n g the l a t t e r p a r t o f t h e S2 t o S3 stages.

A s a r e s u l t , the secondary

shows a heterogeneous d i s t r i b u t i o n o f p o l y s a c c h a r i d e s .

wall

Lignin deposition

lags b e h i n d the p o l y s a c c h a r i d e f o r m a t i o n . T h e m o s t s t r i k i n g fact is t h a t the m o n o l i g n o l s , w h i c h are synthesized i n the c y t o p l a s m a n d secreted t o t h e i n n e r surface o f the n e w l y f o r m e d cell w a l l , pass t h r o u g h the p r e - e x i s t i n g cell w a l l a n d reach the sites where l i g n i f i c a t i o n is p r o c e e d i n g . T h e r e i s , however, no i n f o r m a t i o n as t o h o w t h i s t r a n s p o r t a c t u a l l y occurs.

Moreover, the

i n t e r m o l e c u l a r r e l a t i o n s h i p s between cellulose, hemicelluloses a n d l i g n i n i n the cell w a l l are s t i l l u n c l e a r . O u r electron m i c r o s c o p y observations have revealed some o f the roles o f cell organellae i n v o l v e d i n biosynthesis o f cell w a l l c o m p o n e n t s :

(i) t h e

p l a s m a m e m b r a n e is the site o f cellulose synthesis. T h i s s u p p o r t s the p r o p o s a l t h a t t e r m i n a l a n d rosette complexes at t h e p l a s m a m e m b r a n e are responsible for cellulose synthesis, ( i i ) T h e G o l g i - b o d i e s a n d s m a l l c i r c u l a r vesicles d e r i v e d f r o m the r - E R ' s are i n v o l v e d i n t h e b i o s y n t h e s i s a n d / o r t r a n s p o r t o f the hemicelluloses.

O u r i n v e s t i g a t i o n s , however, c o u l d n o t

d i s t i n g u i s h between w h a t t y p e o f cell organellae c o n t a i n e d w h a t k i n d o f hemicelluloses, a n d h o w these p o l y m e r s were processed i n t h e o r g a n e l l a e . (iii) T h e G o l g i - b o d i e s a n d s - E R ' s p a r t i c i p a t e i n the biosynthesis a n d / o r t r a n s p o r t o f m o n o l i g n o l s . It is expected t h a t new techniques o f b o t h elect r o n m i c r o s c o p y a n d b i o c h e m i s t r y w i l l i m p r o v e o u r knowledge o f the precise sites where e n z y m a t i c reactions l e a d i n g t o l i g n i n f o r m a t i o n o c c u r . Literature Cited

1. Meier, H.; Wilkie, K. C. B. Holzforschung 1959, 13, 177. 2. Meier, H. J. Polym. Sci. 1961, 51, 11. 3. Côté, W. Α., Jr.; Kutscha, N. P.; Simon, B. W.; Timell, T. E. Tappi 1968, 51, 33. 4. Larson, P. R. Holzforschung 1969, 23, 17. 5. Larson, P. R. Tappi 1969, 52, 2170. 6. Hardell, H.-L.; Westermark, U. Proc. 1st Int. Symp. Wood Pulping Chem. 1981, I:32. 7. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1983, 29, 183. 8. Thiéry, J. J. Microscopie 1967, 6, 987. 9. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1984, 30, 103. 10. Wardrop, A. B. Tappi 1957, 40, 225.

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11. Imagawa, H.; Fukazawa, K.; Ishida, S. Res. Bull. Coll. Exp. Forests, Hokkaido Univ. 1976, 33, 127. 12. Fujita, M.; Saiki, H.; Harada, H. Mokuzai Gakkaishi 1978, 24, 158. 13. Wardrop, A. B. In Lignins; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971; p. 19. 14. Wardrop, A. B. Appl. Poly. Symp. 1976, 28, 1041. 15. Kutscha, N. P.; Schwarzmann, J. M. Holzforschung 1975, 29, 79. 16. Kishi, K.; Harada, H.; Saiki, H. Bull. Kyoto Univ. Forests 1982, 54, 209. 17. Saka, S.; Thomas, R. J. Wood Sci. Technol. 1982, 16, 167. 18. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1981, 27, 813. 19. Takabe, K. Ph.D. Thesis, Kyoto University, Kyoto, 1984. 20. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Res. Bull. Coll. Exp. Forests, Hokkaido Univ. 1986, 43, 783. 21. Brown, R. M., Jr.; Haigler, C. H.; Suttie, J.; White, A. R.; Roberts, E.; Smith, C.; Itoh, T.; Cooper, K. J. Appl. Polym. Sci. (Appl. Polym. Symp.) 1983, 37, 33. 22. Takabe, K.; Harada, H. Mokuzai Gakkaishi 1986, 32, 763. 23. Sugiyama, J.; Otsuka, Y.; Murase, H.; Harada, H. Holzforschung 1986, 40(Suppl.), 31. 24. Pickett-Heaps, J. D. J. Cell. Sci. 1968, 3, 55. 25. Fowke, L. C.; Pickett-Heaps, J. D. Protoplasma 1972, 74, 19. 26. Ryser, U. Protoplasma 1979, 98, 223. 27. Sugiyama, J.; Harada, H. Mokuzai Gakkaishi 1986, 32, 770. 28. Pickett-Heaps, J. D. Protoplasma 1968, 65, 181. 29. Fujita, M.; Harada, H. Mokuzai Gakkaishi 1979, 25, 89. 30. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1985, 31, 613. RECEIVED May 19, 1989

Plant Cell Wall Polymers

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